KR101947702B1 - Human monoclonal anti-pd-l1 antibodies and methods of use - Google Patents

Abstract

The present invention includes human monoclonal antibodies that bind to PD-L1 (also known as prognostic cell death 1 ligand 1 or B7H1). Binding of the antibody of the present invention to PD-L1 can be used to inhibit binding and ligand-mediated activity to its receptor PD1 (Prospectus 1) and to treat cancer and chronic viral infections.

Description

Human monoclonal anti-PD-L1 antibodies and methods of use < RTI ID = 0.0 > {HUMAN MONOCLONAL ANTI-PD-Ll ANTIBODIES AND METHODS OF USE}

Related application

This application claims priority to U.S. Provisional Application No. 61 / 709,731, filed October 4, 2012, and U.S. Provisional Application No. 61 / 779,969, filed March 13, 2013, the contents of which are incorporated herein by reference in their entirety .

Field of the Invention

The present invention relates generally to anti-PD-L1 (also known as prognostic cell death-1 ligand 1 or B7H1) antibodies and methods of use thereof.

The immune system must balance between an effective response that eliminates pathogenic integrants and a tolerance to prevent autoimmune disease. T cells are important for maintaining this balance, and their proper regulation is primarily mediated by the B7-CD28 family molecule. The interaction between a B7 family member functioning as a ligand and a CD28 family member functioning as a receptor not only provides an important positive signal that initiates, increases and maintains a T cell response, but also limits the T cell response , ≪ / RTI > terminate and / or attenuate. Members of the CD28 family, termed PD-1 (also known as prognostic cell death -1), are elevated in activated T cells, B cells, and monocytes. PD-1 has two ligands identified in the B7 family, PD-L1 (also known as BH71 or prospective cell death-1 ligand 1) and PD-L2. PD-L1 expression, which is mainly found in activated antigen presenting cells (APC), tends to be more restrictive, whereas PD-L1 expression is expressed in hematopoietic cells (including activated T cells, B cells, monocytes, dendritic cells and macrophages) And peripheral non-lymphoid tissues (including heart, skeleton, muscle, placenta, lung, kidney and liver tissue). Broad expression of PD-L1 plays an important role in regulating PD-1 / PD-L1-mediated peripheral tolerance.

Binding between PD-L1 and PD-1 has a great effect on controlling T cell responses. In particular, PD-L1 / PD-1 interactions inhibit T cell proliferation and the production of effector cytokines, such as IL-2 and IFN-y, that mediate T cell activity and immune responses. These negative regulatory functions are important for preventing T cell-mediated autoimmunity and immunopathology. However, the PD-1 / PD-L1 axis also plays a role in T cell exhaustion, demonstrating that negative regulatory functions inhibit T cell responses to host damage. Long-lasting or chronic antigen stimulation of T cells can induce an immunological feedback mechanism of negative suppression of antigen-specific responses and cause immune evasion of the pathogen. T cell depletion also gradually physically removes the antigen-specific T cell itself. PD-1 expression in T cells is upregulated during chronic antigen stimulation and binding to PD-L1 blocks effector function in both CD4 + (T helper cells) and CD8 + (cytotoxic T lymphocytes or CTL) T cells, PD-1 / PD-L1 interaction T cell depletion.

More recently, it has been shown that some chronic viral infections and cancers develop an immune avoidance strategy that specifically utilizes the PD-1 / PD-L1 axis by inducing PD-1 / PD-L1-mediated T cell depletion. Many human tumor cells and tumor-associated antigen presenting cells express PD-L1 at high levels, indicating that the tumor induces T cell depletion that avoids anti-tumor immune responses. In chronic HIV infection, HIV-specific CD8 + T cells are functionally impaired, resulting in decreased ability to produce cytokine and effector molecules and decreased ability to proliferate. In studies, PD-1 has been shown to be elevated in HIV-specific CD8 + T cells of HIV-infected individuals, suggesting that PD-1 / PD-L1 pathway blockade has therapeutic potential for treatment of HIV infection and AIDS patients Lt; / RTI > Taken together, agents that block the PD-1 / PD-L1 pathway will provide a novel therapeutic approach for various diseases and conditions associated with various cancers, HIV infections, and / or T cell depletion. Accordingly, there is an urgent need for an agent capable of blocking or preventing the PD-1 / PD-L1 interaction.

The present invention is based on the discovery of monoclonal antibodies that bind to PD-L1. Monoclonal antibodies are fully human antibodies. The antibody binds to PD-L1. Antibodies are referred to herein as huPD-L1 antibodies.

PD-L1 is also known as the anticipated cell death 1 ligand 1, the predicted ligand 1, PDCD1 ligand 1, PDCD1L1, PDL1, B7 homologue 1, B7H1, B7-H, CD274 and CD274 antigens.

The present invention provides an isolated humanized monoclonal antibody that binds to human PD-L1 comprising a heavy chain having three CDRs each comprising an amino acid sequence SYGIS (SEQ ID NO: 57), WISAYNGNTNYAQKLED (SEQ ID NO: 70), and ALPSGTILVGGWFDP And light chain having three CDRs each comprising the amino acid sequence TRSSGNIASNYVQ (SEQ ID NO: 101), EDNQRPS (SEQ ID NO: 115), and QSYDSSNLWV (SEQ ID NO: 127); The heavy and amino acid sequences QGDSLRSYYAS (SEQ ID NO: 102), GKNNRPS (SEQ ID NO: 116), and SEQ ID NO: 116 have three CDRs comprising the amino acid sequences SYALS (SEQ ID NO: 58), AISGGGGSTYYADSVKD (SEQ ID NO: 71), and DVFPETFSMNYGMDV And three CDRs each comprising NSRDSSGNHYV (SEQ ID NO: 128); The heavy chain and amino acid sequences GGSDIGRKSVH (SEQ ID NO: 103), SDRDRPS (SEQ ID NO: 117), and SEQ ID NO: 117 having three CDRs each comprising the amino acid sequences DYAMH (SEQ ID NO: 60), LISGDGGSTYYADSVKD (SEQ ID NO: 73), and VLLPCSSTSCYGSVGAFDI And QVWDNNSDHYV (SEQ ID NO: 129), respectively; The heavy chain and amino acid sequences TGTSSDVGGYNYVS (SEQ ID NO: 104), DVSNRPS (SEQ ID NO: 118), and SEQ ID NO: 118 having three CDRs each comprising the amino acid sequence NYDMS (SEQ ID NO: 61), RVNWNGGSTTYADAVKD (SEQ ID NO: 74), and EFVGAYDL And SSYTSSTLP (SEQ ID NO: 130), respectively; The heavy and amino acid sequences RASQSIGNSLA (SEQ ID NO: 105), GASSRAT (SEQ ID NO: 119), and SEQ ID NO: 90 have three CDRs each comprising the amino acid sequences GLYIH (SEQ ID NO: 62), WIIPIFGTANYAQKFED (SEQ ID NO: 75), and GLRWGIWGWFDP And QQHTIPTFS (SEQ ID NO: 131), respectively; The heavy and amino acid sequences RASQGIGSYLA (SEQ ID NO: 106), AASTLQS (SEQ ID NO: 120), and SEQ ID NO: 120 having three CDRs comprising the amino acid sequences DNAIS (SEQ ID NO: 63), WIIPIFGKPNYAQKFED (SEQ ID NO: 76), and TMVRGFLGVMDV And QQLNNYPIT (SEQ ID NO: 132), respectively; The heavy and amino acid sequences SGDKLGNKYAY (SEQ ID NO: 107), QDIKRPS (SEQ ID NO: 121), and SEQ ID NO: 12 having three CDRs each comprising the amino acid sequence SYAMS (SEQ ID NO: 64), AISGSGGSTYYADSVKD (SEQ ID NO: 77), and DQFVTIFGVPRYGMDV And QTWDNSVV (SEQ ID NO: 133), respectively; The heavy and amino acid sequences TRSSGSIDSNYVQ (SEQ ID NO: 108), EDNQRPS (SEQ ID NO: 115), and SEQ ID NO: 115, which have three CDRs comprising the amino acid sequences SYAIS (SEQ ID NO: 57), WIIPIFGTANYAQKFED (SEQ ID NO: 78), and GRQMFGAGIDF And QSYDSNNRHVI (SEQ ID NO: 134), respectively; The heavy and amino acid sequences TRSSGNIGTNYVQ (SEQ ID NO: 109), EDYRRPS (SEQ ID NO: 122), and SEQ ID NO: 122, which have three CDRs each comprising the amino acid sequences TYALN (SEQ ID NO: 65), RIVPLIGLVNYAHNFED (SEQ ID NO: 79), and EVYGGNSDY And QSYHSSGWE (SEQ ID NO: 135), respectively; The heavy and amino acid sequences GGNNIGSKGVH (SEQ ID NO: 110), DDSDRPS (SEQ ID NO: 123), and SEQ ID NO: 123 with three CDRs each comprising the amino acid sequences SHGIT (SEQ ID NO: 66), WISAHNGHASNAQKVED (SEQ ID NO: 80), and VHAALYYGMDV And QVWDSSSDHWV (SEQ ID NO: 136), respectively; The heavy and amino acid sequences TRSSGSIASNYVQ (SEQ ID NO: 111), EDNQRPS (SEQ ID NO: 115), and SEQ ID NO: 115 have three CDRs each comprising the amino acid sequences RHGMH (SEQ ID NO: 67), VISHDGSVKYYADSMKD (SEQ ID NO: 81), and GLSYQVSGWFDP And QSYDSTTPSV (SEQ ID NO: 137), respectively; The heavy and amino acid sequences TRSSGSIASNYVQ (SEQ ID NO: 112), EDNQRPS (SEQ ID NO: 115), and the heavy and light chain amino acid sequences SYSGS (SEQ ID NO: 58), WTSPHNGLTAFAQILED (SEQ ID NO: 82), and VHPVFSYALDV And QSYDGITVI (SEQ ID NO: 138), respectively; The heavy and amino acid sequences TRSSGSIASHYVQ (SEQ ID NO: 113), EDNKRPS (SEQ ID NO: 124), and SEQ ID NO: 128, having three CDRs each comprising the amino acid sequences TYAFS (SEQ ID NO: 68), RIIPILGIANYAQKFED (SEQ ID NO: 83), and DGYGSDPVL And QSYDSSNRWV (SEQ ID NO: 139), respectively; (SEQ ID NO: 114), YKSDSNKQQAS (SEQ ID NO: 125) with three CDRs each comprising the amino acid sequence NYGIS (SEQ ID NO: 69), WISAYNGNTNYAQKVED (SEQ ID NO: 84), and GDFRKPFDY , And MIWYSSAVV (SEQ ID NO: 140), respectively.

In one aspect, the antibody is monovalent or divalent. In another aspect, the antibody is a single chain antibody.

The invention provides a V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 1 and SEQ ID NO: 3; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 5 and SEQ ID NO: 7; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 9 and a V _L nucleotide sequence comprising SEQ ID NO: 11; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 13 and a V _L nucleotide sequence comprising SEQ ID NO: 15; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 17 and a V _L nucleotide sequence comprising SEQ ID NO: 19; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 21 and a V _L nucleotide sequence comprising SEQ ID NO: 23; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 25 and a V _L nucleotide sequence comprising SEQ ID NO: 27; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 29 and a V _L nucleotide sequence comprising SEQ ID NO: 31; A V _H nucleotide sequence comprising SEQ ID NO: 33 and a V _L nucleotide sequence comprising SEQ ID NO: 35; A V _L nucleotide sequence comprising the V _H nucleotide sequence comprising SEQ ID NO: 37 and SEQ ID NO: 39; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 41 and a V _L nucleotide sequence comprising SEQ ID NO: 43; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 45 and a V _L nucleotide sequence comprising SEQ ID NO: 47; A V _L nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 49 and a V _L nucleotide sequence comprising SEQ ID NO: 51; Or a V _H nucleotide sequence comprising SEQ ID NO: 53 and a V _L nucleotide sequence comprising SEQ ID NO: 55.

In another aspect, the invention provides a V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 2 and a V _L amino acid sequence comprising SEQ ID NO: 4; A V _H amino acid sequence comprising SEQ ID NO: 6 and a V _L amino acid sequence comprising SEQ ID NO: 8; A V _H amino acid sequence comprising SEQ ID NO: 10 and a V _L amino acid sequence comprising SEQ ID NO: 12; A V _H amino acid sequence comprising SEQ ID NO: 14 and a V _L amino acid sequence comprising SEQ ID NO: 16; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 18 and SEQ ID NO: 20; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 22 and SEQ ID NO: 24; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 26 and SEQ ID NO: 28; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 30 and a V _L amino acid sequence comprising SEQ ID NO: 32; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 34 and SEQ ID NO: 36; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 38 and SEQ ID NO: 40; A V _H amino acid sequence comprising SEQ ID NO: 42 and a V _L amino acid sequence comprising SEQ ID NO: 44; A V _H amino acid sequence comprising SEQ ID NO: 46 and a V _L amino acid sequence comprising SEQ ID NO: 48; A V _L amino acid sequence comprising a V _H amino acid sequence comprising SEQ ID NO: 50 and a V _L amino acid sequence comprising SEQ ID NO: 52; Or a V _H amino acid sequence comprising SEQ ID NO: 54 and a V _L amino acid sequence comprising SEQ ID NO: 56.

In some aspects, the antibody has a binding affinity within the range of 10 ^-5 M to 10 ^-12 M.

In another aspect, the antibody is also a bispecific antibody that binds to a tumor-associated antigen, cytokine or cell surface receptor. For example, the tumor-associated antigen is CAIX. For example, the cytokine is IL-10. For example, cell surface receptors are CCR4, IL21R, BTLA, HVEM or TIM3.

The present invention provides an antibody that is conjugated to a therapeutic agent. For example, the therapeutic agent is a toxin, radioactive label, siRNA, small molecule, or cytokine.

The present invention provides cells that produce any of the above antibodies.

The present invention also provides a method of selectively killing tumor cells, comprising contacting said cells with any of said antibodies. In one aspect, selective killing occurs by antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and antibody dependent cellular killing (ADCP). In another aspect, the tumor cells express PD-L1.

The present invention also provides a method of preventing or reversing T cell depletion comprising administering to a subject in need thereof a composition comprising any of the above antibodies.

The present invention also provides a method of increasing an immune response to an antigen comprising administering to a subject in need thereof a composition comprising any of the antibodies. In one aspect, the antigen is a viral antigen, a bacterial antigen, or a tumor-associated antigen. In another aspect, the virus antigen is HIV. In another aspect, the tumor-associated antigen is CAIX. In another aspect, the antibody is administered before or after exposure to the antigen. In another aspect, administration of the antibody increases antigen-specific T cell activity. In another aspect, the T cell is an effector T cell.

The present invention also provides a method for treating or alleviating a cancer symptom, comprising administering to a subject in need thereof a composition comprising any of the above antibodies. For example, cancer is renal cell cancer or breast cancer. For example, cancer is cancer in which PD-L1 is overexpressed. In another embodiment, the cancer is a cancer that induces T cell depletion.

The present invention also provides a method for treating or ameliorating a symptom of chronic viral infection comprising administering to a subject in need thereof a composition comprising any of the above antibodies. For example, a chronic viral infection is an HIV infection. For example, chronic viral infection is a viral infection that leads to T cell depletion.

The present invention relates to a pharmaceutical composition comprising at least one compound selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, , 47, 49, 51, 53 or 55, respectively.

In another aspect, the present invention provides a method of screening for a compound of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 42, 44, 46, 48, 50, 52, 54 or 56 of SEQ ID NO: 2.

In another aspect, the present invention provides a method of screening for a compound of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 42, 44, 46, 48, 50, 52, 54 or 56 amino acids.

In yet another aspect, the present invention relates to a method for the treatment of cancer, comprising administering a therapeutically effective amount of a compound of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 41, 43, 45, 47, 49, 51, 53 or 55. [ The present invention provides a pharmaceutical composition comprising at least one compound selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, , 48, 50, 52, 54, or 56, respectively. The invention also provides a cell comprising any of said vectors.

In any of the methods of the disclosure, the route of administration may be parenteral (e.g., intravenously), intradermally, subcutaneously, orally (e.g., by inhalation) Topical), transmucosal, and rectal administration.

In any of the methods of the disclosure, the subject is, for example, a mammal. The mammal is, for example, a human.

Other features and advantages of the invention will be apparent from and elucidated in the following specification and claims.

Figure 1: Amino acid sequence of anti-PD-L1 scFv-phage clone (14 clones). Framework regions 1-4 (FW1-4), complementarity determining regions 1-3 (CDR1-3), and family names of IGHV and IGLV / IGKV. Kabat numbers are used. Symbol description: "." AA is a common sequence, "X" is a non-common AA, and "-" is a blank (ie, no AA).
2] Analysis of binding of human PD-L1 (hPD-L1) expressing cells and huPD-L1 antibody by FACS. Four types of cells were tested, including parental cell line 300.9, and 300.9 cells transfected with hPD-L1, hPD-L2 or the human C-type lectin domain family 2 member (hCLEC2D). GF1538 is a humanized Ab to hPD-L1. GF1757 is a humanized Ab to hPD-L2. The secondary antibody is PE-goat anti-human IgG.
[Figure 3] In a competitive FACS analysis Inhibition of hPD-1 binding to hPD-L1 by anti-PD-L1 phage-antibody. All anti-hPD-L1 Ab in the form of phage-scFv were tested for inhibition of binding of hPD-L1 expressing 293T cells to hPDl-hFc fusion protein. 10 ¹² pfu of phage-scFv were mixed with ~ 0.25 μg / ml of soluble hPD1-hFc and added to 293T cells transfected with the hPD-L1 expression plasmid. After washing, cells were incubated with FITC-anti-human IgG antibody to measure hPDl-hFc binding to hPD-L1 on the cell surface.
[Figure 4] In a competitive FACS analysis Inhibition of hPD-1 binding to hPD-L1 by anti-PD-L1 soluble antibodies. All anti-hPD-L1 Ab were preincubated with 300.9 cells transfected with hPD-L1 expression plasmid for 30 minutes at indicated concentrations, then 0.125 ug hPD-1-mouse IgG2a was added to each reaction and further incubated for 30 minutes Lt; / RTI > After washing, PE-chlorine-anti-mouse IgG2a Ab was added, followed by washing and FACS analysis. GF1538 is a humanized Ab to hPD-L1. GF1757 is a humanized Ab to hPD-L2.
Figure 5: Design and formation of bispecific antibodies.
Fig. 6: Determination of bispecific antibody (bsAb) structure. A) Schematic showing the " grip on the hole " approach that binds the CH3 domain with a bispecific antibody recognizing CAIX and PD-L1. B) Schematic representation of three types of bsAb constructs with different mutations in the CH2 domain that alter ADCC activity.
[Figure 7] Generation of bispecific antibodies and their functions. A) Protein showing dissociation of (G37 KIHA) antibody engineered under reducing conditions compared to conjugated (unreduced) control IgG, parental G37 (WT), and bispecific (G37 KIHA + PD-L1 KIHB) Gel. B) Protein gel showing dissociation of (PD-L1 KIHB) antibody engineered under reducing conditions as compared to control IgG, parental PDL-1 (WT), and bispecific (G37 KIHA + PD-L1 KIHB) antibodies. C) Analysis of bispecific antibody binding to CAIX ⁺ PDL-1 ^- SKRC-52 cells by flow cytometry.
[Fig. 8] Investigation of functional characteristics of PD-L1 specific mAb42. PBMCs from four healthy donors (D1-D4) were cultured in the presence of αPDL1 (mAb42) or a control allogeneic antibody, stimulated with 0.1 μg / ml SEB for 48 hours, and TNFα production was measured in MSD units. The data The mean ^* , p < 0.0005, of the triplicate experiments.

The present invention provides a humanized monoclonal antibody specific for PD-L1, also known as B7H1. Antibodies were identified by phage display antibody library selection using protein liposome-bound-PD-L1 as library selection target. This antibody represents a new class of human monoclonal antibodies to PD-L1.

The anti-PD-L1 human monoclonal antibody is referred to herein as " huPD-L1 antibody ".

The binding of PD-L1 to PD-1 negatively regulates T cell antigen-specific responses important for tolerance and prevention of autoimmunity and immunopathology. However, excessive PD-L1 / PD-1 interaction, which may be induced by chronic antigen stimulation, may result in inhibition of T cell antigen-specific responses and loss of T cells, which are characteristic of T cell depletion. T cell depletion is a condition of T cell dysfunction that can occur in chronic infections and cancer. It is defined as poor effector function, persistent expression of inhibitory receptors, and transcriptional state different from functional effector or memory T cells. Depletion interferes with infection and management of tumor progression.

PD-L1 overexpression was detected in various cancers. For example, in breast cancer, PD-L1 is over-expressed and associated with high-risk prognostic factors. In renal cell carcinoma, PD-L1 is elevated and increased expression of PD-1 has also been identified in tumor invasive white blood cells. Anti-PD-L1 and anti-PD-1 antibodies demonstrated some clinical efficacy in the first stage of renal cell carcinoma. Therapeutic agents capable of binding PD-I or PD-L1 may be useful for specifically targeting tumor cells. Agents that are capable of blocking PD-1 / PD-L1 interactions may be more useful in treating cancers that lead to T cell depletion that avoids anti-tumor T cell activity. Use of the agent alone or in combination with other anti-cancer therapies can effectively target the tumor cells overexpressing PD-L1, increase anti-tumor T cell activity, and enhance the immune response targeting tumor cells.

PD-1 and PD-L1 may also be up-regulated by T cells after chronic antigen stimulation, e. G. Chronic infection. In chronic HIV infection, HIV-specific CD8 + T cells are functionally impaired, resulting in decreased production capacity of cytokine and effector molecules and decreased proliferative capacity. PD-1 is highly expressed in HIV-specific CD8 + T cells of HIV-infected individuals. Thus, blocking of the pathway may increase the immune response to HIV by increasing the ability of HIV-specific cells to proliferate and produce cytokines in response to HIV peptide stimulation. Other chronic infections, such as chronic viral, bacterial or parasitic infections, may also benefit from the use of PD-1 / PD-L1 blockers.

The present invention provides a human monoclonal antibody that specifically binds to a PD-L1 protein. The antibody binding of the present invention to PD-L1 hinders the ability of the ligand to bind its receptor PDl. By a variety of mechanisms, huPD-L1 antibodies prevent negative feedback mechanisms that inhibit T cell responses. In some cases, huPD-L1 antibodies prevent, arrest, or reverse T cell depletion. Administration of huPD-L1 antibody may result in an increase in T cell proliferation, an increase in antigen-specific T cell activity, and an increase in the production of effector cytokines. In some cases, the huPD-L1 antibody promotes or augments the antigen-specific immune response. The immune response may be mediated by effector T cells.

The huPD-L1 antibody is monovalent or divalent and includes short or double chains. Functionally, the binding affinity of the huPD-L1 antibody is within the range of 10 ^-5 M to 10 ^-12 M. For instance, the combination of huPD-L1 antibody affinity is 10 ^-6 M to 10 ^-12 M, 10 ^-7 M to 10 ^-12 M, 10 ^-8 M to 10 ^-12 M, 10 ^-9 M to 10 ^-12 M, 10 ^-5 M to 10 ^-11 M, 10 ^-6 M to 10 ^-11 M, 10 ^-7 M to 10 ^-11 M, 10 ^-8 M to 10 ^-11 M, 10 ^-9 M to 10 ^-11 M, 10 ^-10 M to 10 ^-11 M, 10 ^-5 M to 10 ^-10 M, 10 ^-6 M to 10 ^-10 M, 10 ^-7 M to 10 ^-10 M, ^10-8 M to 10 ^-10 M, 10 ^-9 M to 10 ^-10 M, 10 ^-5 M to 10 ^-9 M, 10 ^-6 M to 10 ^-9 M, 10 ^-7 M to 10 ^-9 M, ^10-8 M to ^10-9 M, 10 ^-5 M to 10 ^-8 M, 10 ^-6 M to 10 ^-8 M, 10 ^-7 M to 10 ^-8 M, 10 ^-5 M to 10 ^-7 M, 10 ^-6 M to 10 ^-7 M or 10 < ^-5 > M to 10 < ^-6 >

In addition, the antibodies of the invention include, but are not limited to, therapeutic agents including toxins, radiolabeled, siRNA or cytokines.

huPD-L1 antibody can induce apoptosis. Cell death is induced by direct or indirect mechanisms. For example, PD-Ll binding by huPD-L1 antibodies can cause complement dependent cytotoxicity (CDC). Alternatively, the huPD-L1 antibody binds to PD-L1 and causes the mobilization of a secondary cell type to kill the PD-L1-expressing target cells. Representative mechanisms by which huPD-L1 antibodies mediate apoptosis by the mobilization of secondary cell types include, but are not limited to, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cellular cytotoxicity (ADCP). Target PD-L1-expressing cell types include tumors and T cells, such as activated T cells.

14 unique Monoclonal huPD-L1 antibody was identified. Ab-18, Ab-22, Ab-30, Ab-31, Ab-32, Ab-38, Ab-42, Ab- Ab-56 and Ab-65.

Nucleic acid and amino acid sequences of monoclonal huPD-L1 antibodies are provided below.

The amino acid sequences of the complementarity determining regions of the heavy and light chains of the huPD-L1 antibody are shown in Tables 15 and 15B below.

The huPD-L1 antibody described herein binds to PD-L1. In one aspect, the huPD-L1 antibody has high affinity and high specificity for PD-L1. In another aspect, the huPD-L1 antibody binds to the PD-1 receptor and can prevent, inhibit or block the ligand PD-L1 binding to its receptor PD-1. In some cases, the huPD-L1 antibody may have some cross-reactivity with PD-L2. In some cases, the huPD-L1 antibody does not exhibit any cross-reactivity with PD-L2. In some cases, huPD-L1 antibodies bind PD-L1 with higher affinity and / or higher specificity than PD-L2.

In addition, the invention features an antibody having the specified percentage identity or similarity to the amino acid or nucleotide sequence of the huPD-L1 antibody described herein. For example, an antibody may be present at a concentration of 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99%, or higher identity. Sequence identity or similarity of the nucleic acids and proteins of the present invention can be determined by sequence comparison and / or alignment by methods known in the art. For example, the percent sequence identity or similarity of a nucleic acid or protein of the present invention can be determined using a sequence comparison algorithm (i.e. BLAST or BLAST 2.0), manual alignment, visual inspection.

With respect to the amino acid sequence, those skilled in the art will appreciate that each substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, protein sequence that alters, adds, deletes, or substitutes a single amino acid or a lower ratio of amino acids in the sequence being coded Quot; conservatively modified variants " as a whole. In some embodiments, the alteration can result in substitution of amino acids with chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are known in the art. This conservatively modified variant of the huPD-L1 antibody can exhibit increased cross-reactivity to PD-L2 as compared to the unmodified huPD-L1 antibody.

Antibody

The term " antibody " as used herein refers to a molecule comprising an immunologically active region of an immunoglobulin molecule and an immunoglobulin (Ig) molecule, i.e., an antigen binding site that specifically binds (immunoreacts) with an antigen . By " specifically binding " or " immunoreactive " is meant that the antibody reacts with one or more antigenic determinant of the desired antigen and does not react with other polypeptides. The antibody may be a polyclonal, monoclonal, chimeric, dAb (domain antibody), short chain, F _ab , F _{ab '} And F _{(ab ') 2} fragments, scFv, and F _ab expression libraries.

A short chain Fv ("scFv") polypeptide molecule is a covalently linked V _H : V _L heterodimer, which can be expressed from a gene fusion comprising V _H - and V _L - coding genes linked by a peptide coding linker (Huston et al. (1988) Proc Nat Acad Sci USA 85 (16): 5879-5883). Numerous methods for identifying chemical structures to convert naturally aggregated but chemically separated light and heavy chain polypeptides of the antibody V region to scFv molecules that can be folded into a three-dimensional structure substantially similar to that of the antigen-binding site are disclosed Has come. See, for example, U.S. Patent Nos. 5,091,513, 5,132,405, 4,946,778.

A very large noncontacted human scFv library has been generated and can be generated to provide a rearranged antibody gene at various sources for an excess of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease specific antibodies (Barbas et al., Proc. Natl. Acad. Sci. USA 89: 9339-43 (1992)); (Zebedee et al., Proc Natl Acad Sci USA 89: 3175-79 (1992)).

In general, antibody molecules obtained from humans are associated with any class of IgG, IgM, IgA, IgE and IgD that are distinguished from each other by the nature of the heavy chain present in the molecule. Some classes also include subclasses, such as IgG ₁ , IgG ₂ And so on. In addition, in humans, the light chain may be a kappa chain or a lambda chain. The term " antigen-binding site, " or " binding domain " refers to a portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues in the N-terminal variable ("V") region of the heavy chain ("H") and light chain ("L"). Three very different stretches in the V region of the heavy and light chains, referred to as " hypervariable regions ", are inserted between the more conserved side stretches known as " framework regions, " Thus, the term " FR " refers to amino acid sequences that are found naturally between and between hypervariable regions in immunoglobulins. In the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in a three-dimensional space forming the antigen-binding surface. The antigen binding surface is complementary to the three dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as " complementarity determining regions " or " CDRs. &Quot; The CDRs of the VH and VL regions of the scFv antibody are shown in FIG.

The term " epitope " as used herein includes any immunoglobulin, scFv, or any protein determinant capable of specifically binding to a T cell receptor. Epitopic determinants usually consist of a group of chemically active surface molecules of molecules such as amino acids or sugar chains, and usually have unusual three-dimensional structural features and unusual charge characteristics. For example, an antibody may be generated for the N-terminal or C-terminal peptide of the polypeptide.

As used herein, the terms " immunological binding, " and " immunoconjugate properties " refer to non-covalent interactions of the immune globulin molecule with the immunogenic bands that occur between immunoglobulin specific antigens. The strength or affinity of the immune binding interaction can be expressed by the dissociation constant (K _d ) of the interaction, and the smaller the K _d , the greater the affinity. The immunobinding properties of the selected polypeptides can be quantified using methods known in the art. One such method involves the steps of measuring the rate of antigen-binding site / antigen complex formation and dissociation, which are geometric, which affects the concentration of the complex partner, the affinity of the interaction, It depends on factors. Thus, the "on rate constant" (K _on ) and the "off rate constant" (K _off ) can be determined by calculating the concentration and the actual rate of binding and dissociation (Nature 361 : 186-87 (1993)). The K _off / K _on ratio can nullify any factor not related to affinity and is equal to the dissociation constant K _d (see generally Davies et al. (1990) Annual Rev Biochem 59: 439-473) . Equilibrium binding constants (K _d) is measured by an analysis such as a combination known to those skilled in the art radioligand analysis, or similar analyzes bar ≤10 μM, preferably ≤10nM, more preferably ≤100 is to ≤10nM, most preferably pM to about 1 pM, it is said that the antibody of the present invention binds specifically to the PD-L1 epitope.

The PD-L1 protein or its derivatives, fragments, analogs, homologs or orthologs of the present invention can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components. The PD-L1 protein or derivatives, fragments, analogs, homologues or orthologs of the present invention bound to protein liposomes can be used as immunogens in the generation of antibodies that immunospecifically bind to these protein components.

Those skilled in the art will appreciate that, without undue experimentation, the human monoclonal antibody can determine whether the human monoclonal antibody of the invention prevents binding to PD-Ll, so that the former has the same specificity as the latter. If the human monoclonal antibody being tested compete with the human monoclonal antibody of the invention, as indicated by the reduced binding by the human monoclonal antibody of the invention, the two monoclonal antibodies will bind to the same or a highly related epitope.

Another method for determining whether a human monoclonal antibody has specificity for a human monoclonal antibody of the invention is to add the human monoclonal antibody of the invention to a human monoclonal antibody that has been previously incubated with the normally reactive PD- To determine if the ability of the human monoclonal antibody tested to bind to its PD-L1 is inhibited. When the human monoclonal antibody to be tested is inhibited, it has perhaps the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening of human monoclonal antibodies of the invention can be performed using PD-L1 to determine if the test monoclonal antibody is able to neutralize PD-L1.

A variety of methods known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the protein of the invention, or to derivatives, fragments, analogs, homologs or orthologs thereof (see, for example, (See Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Antibodies can be purified using affinity chromatography using Protein A or Protein G, which primarily provides IgG fractions of the immunized sera, in a known manner. Alternatively or in the alternative, the specific antigen, which is a target of an immunoglobulin or an epitope thereof, can be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. Purification of immunoglobulin is discussed in, for example, D. Wilkinson, The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000) .

The term " monoclonal antibody " or " MAb " or " monoclonal antibody composition " as used herein refers to a population of antibody molecules comprising only a single molecular species of an antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining region (CDR) of a monoclonal antibody is the same in all molecules of a population. The MAb comprises an antigen binding site capable of immunoreacting with a specific epitope of an antigen characterized by a unique binding affinity thereto.

Monoclonal antibodies can be prepared using the hybridoma method, for example, the method described in Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to induce a lymphocyte capable of producing or producing an antibody that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will typically comprise a protein antigen, a fragment thereof, or a fusion protein thereof. In general, peripheral blood lymphocytes are used when cells of human origin are preferred, or spleen cells or lymph node cells when a non-human mammalian source is preferred. Lymphocytes are then fused with infinite proliferating cell lines using suitable fluxes such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice , Academic Press, (1986) pp. 59-103) ). Infinitely proliferating cell lines are typically transformed mammalian cells, particularly murine, bovine and human derived myeloma cells. Usually, a myeloma cell line is used in rats or mice. The hybridoma cells may preferably be cultured in a suitable culture medium comprising one or more substances that inhibit the growth or survival of unfused infinitely proliferating cells. For example, when the parental cell lacks the enzyme hypoxanthine guanine phospholibosyl transferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, which is a substance that prevents the growth of HGPRT deficient cells , And thymidine (" HAT medium ").

Preferred infinite proliferating cell lines are those that efficiently fuse, support stable and high levels of antibody expression by selected antibody producing cells, and are sensitive to media, such as HAT media. More preferred infinite proliferating cell lines are mouse myeloma cell lines and they are obtained from, for example, the Salk Institute Cell Distribution Center (San Diego, Calif.) And the American Type Culture Collection (Manassas, Virginia) . In addition, human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

Cultured cells in which the hybridoma cells are cultured can then be assayed for the presence of a monoclonal antibody directed against the antigen. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cell can be determined by immunoprecipitation or in vitro binding assay, for example, radioimmunoassay (RIA) or enzyme linked immunosorbant assay (ELISA) . Such techniques and assays are well known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of the literature (Munson and Pollard, Anal. Biochem., 107: 220 (1980)). In therapeutic applications of monoclonal antibodies, it is also important to identify antibodies with high degree of specificity and high binding affinity for the target antigen.

Once the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice , Academic Press, (1986) pp. 59-103) ). Suitable culture media for this purpose include, for example, DMEM (Dulbecco's Modified Eagle's Medium) and RPMI-1640 medium. Alternatively, the hybridoma cells can be cultured in vivo, such as a plurality of mammals.

The monoclonal antibody secreted by the subclone can be isolated from the culture medium or from a plurality of solutions by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose hydroxylapatite chromatography, gel electrophoresis, dialysis, Can be isolated or purified.

In addition, monoclonal antibodies can be produced by recombinant DNA methods, for example, the methods described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily isolated using conventional methods (for example, using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy chain and the light chain of a mouse antibody) Can be analyzed. The hybridoma cells of the present invention serve as a preferred source of the DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into host cells, such as monkey COS cells, Chinese hamster ovary cell (CHO) cells, or myeloma cells that do not produce the original immunoglobulin protein Monoclonal antibodies can be synthesized in recombinant host cells. Also, the DNA may be substituted with the coding sequence of the human heavy and light chain constant domains in place of the homologous mouse sequence (see U.S. Patent No. 4,816,567 and Morrison, Nature 368, 812-13 (1994)), the immunoglobulin coding sequence By covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide. The non-immunoglobulin polypeptide may be substituted into the antibody constant domain of the present invention to produce chimera 2 antibody, or may be substituted into the variable domain of one antigen binding site of the antibody of the present invention.

Fully human antibodies are antibody molecules in which all sequences of light and heavy chains, including CDRs, are derived from human genes. Such antibodies are referred to herein as " humanized antibodies ", " human antibodies ", or " fully human antibodies ". Human monoclonal antibodies may be used in combination with trioma technology to produce human monoclonal antibodies, human B cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4: 72), and EBV hybridoma technology Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be obtained by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030), or human B cells with the Epstein Barr virus (See Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using another technique including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991)); Marks et al., J Mol. Biol., 222: 581 (1991)). Similarly, a human antibody can be made by introducing the human immunoglobulin genetic locus into a transgenic animal, e. G., A partially or completely inactivated mouse, such as an endogenous immunoglobulin gene. In attack, human antibody production very similar to that seen in humans is observed in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 569,825, 5,625,126, 5,633,425, 5,661,016 and Marks et al., Bio / Technology 10, 779-783 (Fishwild et al, Nature Biotechnology 14, 845-51 (1996)); (Lonberg et al., Nature 368 856-859 (1994)); (Morrison, Nature 368, 812-13 (Neuberger, Nature Biotechnology 14, 826 (1996)) and (Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995)).

Human antibodies can also be produced using transgenic non-human animals that have been modified to produce fully human antibodies, rather than animal endogenous antibodies, in response to attack by antigens (see PCT Publication No. WO 94/02602). The endogenous gene encoding the heavy and light chain immunoglobulins in the non-human host becomes incapacitated, and the active genetic locus encoding the human heavy and light chain immunoglobulins is inserted into the genome of the host. Human genes are incorporated, for example, using yeast artificial genomes containing the necessary human DNA segments. Animals that provide all the desired strains are then obtained with their offspring by interbreeding the intermediate transgenic animals with fewer strains than full complement. A preferred embodiment of the non-human animal is a mouse and is referred to as Xenomouse ( ^TM ) as disclosed in PCT publications WO 96/33735 and WO 96/34096. The animal produces B cells that secrete fully human immunoglobulin. Antibodies can be obtained, for example, from infinitely proliferating B cells derived from animals, such as hybridomas, obtained directly from animals following immunization with an immunogen of interest as a preparation of polyclonal antibodies, or alternatively producing monoclonal antibodies . In addition, a gene encoding an immunoglobulin having a human variable region can be recovered and expressed to directly obtain an antibody, or can be further modified to obtain an antibody analogue such as, for example, a short chain Fv (scFv) molecule.

An example of a method for producing a non-human host typified by a mouse lacking the expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. This is accomplished by targeting a J segment gene from one or more endogenous heavy chain gene locus in embryonic stem cells to prevent the rearrangement of the genetic loci and prevent the formation of transcripts of the rearranged immunoglobulin heavy chain locus locus, Removing by a vector; And producing somatic cells and germ cells from embryonic stem cells with a transgenic mouse comprising a selectable marker coding gene.

One method of producing antibodies of interest, such as human antibodies, is disclosed in U.S. Patent No. 5,916,771. The method comprises the steps of introducing an expression vector comprising a nucleotide sequence encoding a heavy chain into a mammalian host cell in a culture, introducing an expression vector comprising a nucleotide sequence encoding a light chain into another mammalian host cell , And fusing the two cells to form hybrid cells. Hybrid cells express antibodies comprising heavy and light chains.

In a further refinement of the method, a method for identifying a clinically relevant epitope on an immunogen, and an associated method for screening for an antibody that immunospecifically binds to an associated epitope with a high affinity, is disclosed in PCT publication WO 99/53049 Lt; / RTI >

The antibody may be expressed by a vector comprising a DNA segment encoding the single chain antibody described above.

These may include vectors, liposomes, naked DNA, adjuvant assisted DNA, gene guns, catheters, and the like. The vector may be a chemical conjugate having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine) as described in WO 93/64701, a viral vector (e. G. PCT / US95 / 02140 (WO 95/22618) which is a fusion protein comprising a targeting moiety (e. G., An antibody specific for a target cell) and a nucleic acid binding moiety (e. G. Protamine) Fusion proteins, plasmids, phages, etc., as described. The vector may be a chromosome, a non-chromosome, or a synthetic vector.

Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Molloy rodent leukemia virus. DNA virus vectors are preferred. Such vectors include, but are not limited to, Fox vectors such as orthotox or avian Fox vectors, herpes virus vectors such as herpes simplex I virus (HSV) vectors (Geller, AI et al., J. Neurochem, 64: 487 (Geller, AI et al., Proc. Natl., (1995)); (Lim, F., et al., In DNA Cloning: Mammalian Systems, D. Glover, Acad. Sci. USA 90: 7603 (1993)); adenoviral vectors (see LeGal LaSalle et al., Proc Natl Acad Sci USA 87: (Yang, et al., J. Virol. 69: 2004 (1995)), which is incorporated herein by reference in its entirety), (Davidson, et al., Nat. Genet 3: 219 ) And adeno-associated viral vectors (see Kaplitt, MG. Et al., Nat. Genet. 8: 148 (1994)).

The poxvirus vector introduces the gene into the cytoplasm of the cell. The avian vox virus vector expresses the nucleic acid only for a short period of time. Adenovirus vectors, adeno-associated viral vectors, and simple herpesvirus (HSV) vectors are preferred for introducing nucleic acids into neutrophils. Adenoviral vectors express for a shorter period of time (about 2 months) than adeno-associated viruses (about 4 months), which is shorter than HSV vectors. The particular vector chosen will depend on the target cell and the condition being treated. The introduction may be by standard techniques, for example infection, transfection, transduction, or transformation. Examples of gene transfer methods include, for example, naked DNA, CaPO ₄ precipitation, DEAE dextran, electroporation, protoplast fusion, lipoic pectin, cell microinjection, and viral vectors.

The vector can basically be used to target any desired target cell. For example, a stereotaxic injection method can be used to send a vector (e. G., Adenovirus, HSV) to a desired location. The particles may also be delivered by intracerebral (icv) injection using a mini pump injection system such as the SynchroMed Infusion System. In addition, methods based on volumetric flow, termed convection, have proven effective in delivering large molecules to the extended region of the brain and can be useful for delivering vectors to target cells (Bobo et al. Proc Natl Acad Sci USA 91: 2076-2080 (1994)) (Morrison et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that may be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injection and oral or other known routes of administration.

Such vectors can be used to express large amounts of antibodies that can be used in a variety of ways. For example, it can be used to detect the presence of PD-L1 in a sample. Antibodies can also be used to bind PD-L1 or try to destroy PD-L1 activity.

Techniques can be tailored to produce single chain antibodies specific for the antigenic proteins of the present invention (see, for example, U.S. Patent No. 4,946,778). In addition, the method F _ab Monoclonal < RTI ID = 0.0 > F _ab < / RTI > fragments with the desired specificity for proteins or derivatives, fragments, analogs or homologs thereof (see e.g. Huse, et al., 1989 Science 246: 1275-1281) Can be confirmed quickly and effectively. Antibody fragments comprising an idiotate for protein antigens can be produced by techniques known in the art. These include (i) F _{(ab ') 2} produced by pepsin degradation of the antibody molecule; (ii) an F _ab fragment generated by reducing a disulfide bond of F _{(ab ') 2} fragment; (iii) _Fab fragments generated by treating the antibody molecule with papain and a reducing agent, and (iv) F _v fragments.

Also, heterozygous antibodies are within the scope of the present invention. Heterozygous antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to undesired cells (US Patent No. 4,676,980) and to treat HIV infection (WO 91/00360, WO 92/200373, EP 03089). It is believed that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, an immunotoxin can be produced using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

With regard to effector function, it may be desirable to modify the antibody of the invention to improve the effect of the antibody, for example in treating cancer. For example, a cysteine residue can be introduced into the Fc region to form interchain disulfide bonds in this region. Thus, the resulting homodimeric antibodies enhanced internalization capacity and / or increased complement mediated cell death and antibody dependent cellular cytotoxicity (Caron et al., J. Exp Med., 176: 1191-1195 (1992)) and (Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, antibodies can be engineered to have dual Fc regions, thereby improving complement dissolution and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989) .

 The present invention also encompasses antibodies conjugated to cytotoxic agents such as toxins (e.g., enzyme active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioactive conjugates) Lt; / RTI >

An enzyme-active toxin, or fragment thereof, that can be used includes a diphtheria A chain, a non-binding active fragment of a diphtheria toxin, an exotoxin A chain (derived from Pseudomonas aeruginosa), a lysine A chain, Aleurites fordii protein, Dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia, Inhibitors, curcine, cortin, sapaonaria officinalis inhibitors, gelonin, mitogelin, resticillin, penomycin, enomycin, and tricothecene. Several radioactive nuclear species are available for the production of radiolabeled antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

A conjugate of an antibody and a cytotoxic agent may comprise various amounts of functional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT) , Di-functional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (e.g., di succinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds Diazonium derivatives such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (for example, toluene 2 , 6-diisocyanate), and a bis-active fluorine compound (for example, 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in the literature (Vitetta et al., Science 238: 1098 (1987)). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating radionuclides to antibodies (see WO94 / 11026).

Those skilled in the art will recognize that a wide variety of possible moieties may be attached to the resulting antibody or other molecule of the invention (see, e. G., Conjugate Vaccines, Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989)).

As long as the antibody and other moieties retain their respective activities, the binding can be carried out by any chemical reaction that will bind the two molecules. Such binding may involve many chemical mechanisms, such as covalent bonding, affinity binding, intercalation, coordination and complex formation. However, the preferred bond is a covalent bond. Covalent bonds can be made by direct condensation of the side chains present or by incorporation of external crosslinking molecules. Many bivalent or multivalent linking agents are useful for binding protein molecules such as antibodies of the invention to other molecules. For example, representative coupling agents may include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. (Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984)), which is not intended to be a complete listing of the various classes of coupling agents known in the art, (Jansen et al., Immunological Reviews 62: 185-216 (1982)) and (Vitetta et al., Science 238: 1098 (1987)). Preferred linkers are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44: 201-208 (1984) describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester See also U. S. Patent No. 5,030, 719, which describes the use of halogenated acetylhydrazide derivatives conjugated to antibodies by oligopeptide linkers. Particularly preferred linkers are (i) EDC (1-ethyl-3- Dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl- alpha -methyl- alpha - (2- pyridyl-dithio) -toluene (Pierce Chem. (Ii) SPDP (succinimidyl-6 [3- (2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat. # 21651G) (V) EDC (Pierce Chem. Co., Cat. # 2165-G) and L-cysteine Sulfo-NHS (N-hydroxysuccinimide (Pierc Chem. Co., Cat. # 24510G).

Because the linkers described above contain components with different properties, they induce conjugates with different physicochemical properties. For example, the sulfo-NHS esters of alkyl carboxylates are more stable than the sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers have lower solubility than sulfo-NHS esters. Also, the linker SMPT can form sterically hindered disulfide bonds and increase stability. Disulfide bonds are generally less stable than other bonds. Because disulfide bonds are cleaved in vitro to form conjugates with lower availability. In particular, sulfo-NHS can increase the stability of carbodiimide bonds. When used in conjunction with sulfo-NHS, carbodiimide bonds (e. G. EDC) form esters with higher resistance to hydrolysis than carboimide bond only reactions.

The antibodies disclosed herein may be formulated as immuno-liposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Acad. Sci. USA, 77: 4030 (1980)) and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes having improved cycle times are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through defined pore size filters to produce liposomes with the desired diameter. Fab 'fragments of the antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982).

PD- L1 ≪ / RTI >

The antibody or fragment thereof of the present invention that specifically binds to the PD-L1 protein may be administered for the treatment of cancer or other proliferative disorders. Many cancers overexpress PD-L1, and upregulation of PD-L1 is associated with high-risk prognostic factors. In addition, overexpression of PD-L1 in tumor cells may indicate a mechanism by which tumor cells, for example, induce T cell depletion, thereby avoiding anti-tumor immunity. Such cancers include renal cell carcinoma and breast cancer. Another typical cancer is cancer associated with or utilizing T cell depletion to avoid anti-tumor T cell activity. The use of an antibody of the invention can increase T cell activity or antitumor immune response by increasing the ability of tumor antigen-specific T cells to proliferate and secrete cytokines in response to stimulation of tumor antigen peptides.

The antibody or fragment thereof of the present invention that specifically binds to the PD-L1 protein can be administered for the treatment of chronic infection. Such infections include, for example, viral, bacterial and parasitic infections. A typical chronic viral infection is HIV. In chronic HIV infection, HIV-specific CD8 + T cells are functionally impaired and exhibit a decreased ability to produce cytokines and effector molecules and a decreased ability to proliferate. PD-1 is highly expressed in HIV-specific CD8 + T cells of HIV-infected individuals. The use of the antibodies of the present invention can enhance T cell activity or antiviral immune responses by enhancing the ability of HIV specific T cells to proliferate and produce cytokines in response to HIV peptide stimulation.

Antibodies of the invention, including bispecific, polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents may be used to treat or prevent cancer in a subject, increase vaccine efficiency, or enhance a natural immune response. Antibody agents, preferably those having high specificity and high affinity for their target antigen, will be administered to the subject and will generally have an effect due to the target and its binding. Administration of the antibody may prevent or inhibit or interfere with the activity of the PD-L1 protein.

The antibody of the present invention can induce apoptosis. Cell death is induced by direct or indirect mechanisms. For example, PD-Ll binding by huPD-L1 antibodies can induce complement dependent cytotoxicity (CDC). Alternatively, the huPD-L1 antibody induces the mobilization of secondary cell types that bind to PD-L1 and kill PD-L1-expressing target cells. Typical mechanisms by which huPD-L1 antibodies mediate apoptosis by the mobilization of secondary cell types include, but are not limited to, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cellular cytotoxicity (ADCP). Target PD-L1-expressing cell types include tumors and T cells, such as activated T cells.

The antibody or fragment thereof of the present invention that specifically binds to the PD-L1 protein can be administered in the form of a pharmaceutical composition for the treatment of cancer or chronic infection. The principles and considerations related to the preparation of therapeutic compositions comprising antibodies and guidance in the selection of constituents are given in, for example, Remington: The Science and Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al. , editors Mack Pub. Co., Easton, Pa., 1995); (Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994); And (Petites And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

The therapeutically effective amount of the present invention generally relates to the amount required to achieve a therapeutic purpose. As mentioned above, this may in some cases be a binding interaction between an antibody that interferes with the function of the target and its target antigen. In addition, the amount required for administration will depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which the administered antibody is consumed from the administered subject. A universal range of therapeutically effective doses of the antibodies or antibody fragments of the invention can be from about 0.1 mg / kg body weight to about 50 mg / kg body weight, as non-limiting examples. The universal dosage frequency may range, for example, from twice daily to once a week.

When antibody fragments are used, a minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of the antibody, the peptide molecule can be designed to retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). In addition, the agent may comprise one or more active compounds, if necessary for the particular condition to be treated, preferably those having complementary activities that do not have deleterious effects on each other. Alternatively, or additionally, the composition may include agents that enhance its function, for example, cytotoxic agents, cytokines, chemotherapeutic agents or growth inhibitors. The molecule is suitably present in an amount effective for the desired purpose.

The active ingredients may also be incorporated into microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules, and poly- (methylmethacrylate) microcapsules, respectively, Can be introduced into systems (e. G., Liposomes, albumin microspheres, nanoparticles and nanocapsules), or into macroemulsions.

The formulation used for in vivo administration must be sterile. This is done by filtering through a sterile filter membrane.

Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers comprising an antibody, wherein the matrix is in the form of a shaped body, e. G., Film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol), polyactide (US Patent No. 3,773,919), L- and ethyl γ- -L- copolymer of glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, for example a loop Rhone depot (LUPRON dEPOT ^TM) (lactic acid-glycolic acid copolymer and leuprolide acetate (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules over 100 days, while poly- Some hydrogels release proteins for a shorter period.

The antibody according to the present invention can be used as a preparation for detecting the presence of PD-L1 (or protein or protein fragment thereof) in a sample. Preferably, the antibody comprises a detectable label. The antibody may be a polyclonal, or more preferably a monoclonal. A complete antibody, or a fragment thereof (e.g., F _ab , scFv, or F _{(ab) 2} ) may be used. The term " labeled " in the context of a probe or antibody refers to a direct labeling of a probe or antibody by binding a detectable substance to a probe or antibody (i.e., physical binding) and a probe by reactivity with another indirectly labeled reagent Or indirect labeling of the antibody. Examples of indirect labeling include detection of a primary antibody that can be detected as fluorescently labeled streptavidin using a fluorescently labeled secondary antibody and a biotin with a DNA labeling end-label. The term " biological sample " means to include tissues, cells and biological fluids isolated from the subject, as well as tissues, cells and body fluids present in the subject. Thus, the use of the term " biological sample " includes fractions or components of blood, including serum, plasma or lymph. That is, the detection method of the present invention can be used to detect an analyte mRNA, protein, or genomic DNA in an in vitro and in vivo biological sample. For example, in vitro techniques for analyte mRNA detection include northern hybridization and in situ hybridization. In vitro techniques for analyte protein detection include enzyme linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. In vitro techniques for analyte genomic DNA detection include Southern hybridization. Methods of conducting immunoassays are described, for example, in "Immunoassay", Vol. 42, JR Crowther (Ed.) Human Press, Totowa, NJ, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996); and (Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985) have. In addition, in vivo techniques for the detection of analyte proteins include the step of introducing the labeled anti-analyte protein antibody into the subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques.

Antibodies directed against the PD-L1 protein (or fragments thereof) may be used in conjunction with techniques related to localization and / or quantitation of the PD-L1 protein (e.g., for determining the PD-L1 protein level in a suitable physiological sample, For the purpose of imaging proteins, etc.). In a given embodiment, a PD-L1 protein specific antibody, or derivative, fragment, analog or homologue thereof comprising an antibody-derived antigen binding domain is used as a pharmaceutically active compound (hereinafter referred to as " therapeutic agent ").

The antibodies specific for the PD-L1 protein of the invention can be used to isolate PD-L1 polypeptides by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Antibodies directed against the PD-L1 protein (or fragments thereof) can be used to genuinely monitor protein levels in tissue as part of a clinical testing method, e. G. To determine the efficacy of a given therapeutic regimen. Detection may be facilitated by binding (i.e., physically binding) a detectable substance to the antibody. Examples of detectable substances include various enzymes, complementary molecular chains, fluorescent substances, luminescent substances, bioluminescent substances and radioactive substances. Examples of suitable enzymes include mustard non-peroxidase, alkaline phosphatase, beta -galactosidase, or acetylcholinesterase; Examples of suitable molecular compartments include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; Examples of luminescent materials include luminol; Examples of bioluminescent materials include luciferase, luciferin and equaurin; And examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

Pharmaceutical composition

Antibodies or agents of the invention (referred to herein as " active compounds "), and derivatives, fragments, analogs and homologs thereof, may be included in pharmaceutical compositions suitable for administration. Such compositions typically include an antibody or agent and a pharmaceutically acceptable carrier. The term " pharmaceutically acceptable carrier " as used herein means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents suitable for pharmaceutical administration. Suitable carriers are described in the current edition of Remington ' s Pharmaceutical Sciences, a standard reference in the art, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. The use of such media and preparations in pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, their use in the composition is contemplated. In addition, auxiliary active compounds may be included in the compositions.

The pharmaceutical composition of the present invention can be formulated to suit its desired route of administration. Examples of the route of administration is parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), a transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used in parenteral, intradermal or subcutaneous applications may include the following components; Diluents such as water for injection, physiological saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid (EDTA); Buffers, for example, acetic acid salts, citric acid salts or phosphates, and preparations for tonicity adjustment such as sodium chloride or dextrose. The pH can be adjusted to an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

(Aqueous) or suspension suitable for injectable use and sterile powders for the ready-to-use formulation of sterile injectable solutions or suspensions. For intravenous administration, suitable carriers include physiological saline, purified water, Cremophor EL (TM) (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. The composition must be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or suspension medium comprising water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained by the use of coatings such as lecithin, by the maintenance of the desired particles in the case of suspensions, and by the use of surfactants. The action of the microorganism can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, In many cases, it is preferred to include isotonic agents in the composition, for example, polyhydric alcohols such as sugars, mannitol, sorbitol, sodium chloride. The injectable composition may contain an agent that delays absorption in the composition, for example, aluminum monostearate and gelatin, so that the absorption can last for a long time.

Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound in an appropriate solvent together with one or a combination of the above listed components. If necessary, it can subsequently be sterilized by filtration. In general, dispersions may be prepared by incorporating the active compound into a sterile vehicle, which comprises a basic dispersion medium and the other desired components listed above. In the case of sterile powders for the production of sterile injectable solutions, the process of manufacture is by vacuum drying and lyophilization to produce powders of the active component and any additional desired components from the previously sterile-filtered solution.

Oral compositions generally comprise an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compounds may be combined with excipients or used in the form of tablets, troches, or capsules. In addition, the oral compositions may be formulated so that the compounds in the liquid carrier are applied orally, swished, spit or swallowed May be prepared using a liquid carrier which is used as an oral cleanser. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following components or compounds of similar characteristics: binders such as microcrystalline cellulose, tragacanth rubber or gelatin; Excipients such as starch or lactose; Disintegrants such as alginic acid, Primogel or corn starch; Lubricants such as magnesium stearate or Sterotes; Glidants, such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Flavorings such as peppermint, methyl salicylate or orange flavor.

 For administration by inhalation, the compound is delivered in an aerosol form from a pressure vessel or dispenser, or sprayer containing a suitable propellant, for example, a gas such as carbon dioxide.

It can also be administered systemically by means of a transmucosal or transdermal route. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, surfactants, bile salts, and fuccinic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished through the use of a nasal spray or suppository. For transdermal administration, the active compound may be formulated into a salve, ointment, gel, or cream as is generally known in the art.

The compounds may also be prepared in the form of suppositories for rectal delivery (for example, with conventional suppository bases such as cocoa butter and other glycerides) or in the form of rectal enema.

In one embodiment, the active compound may be formulated into a controlled release formulation, including, for example, an implantable and microencapsulated delivery system, with a carrier to protect the compound from being rapidly removed from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods of making such formulations will be apparent to those skilled in the art. Materials are available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to cells transfected with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example as described in U.S. Patent No. 4,522,811.

The oral or parenteral composition It is particularly advantageous to formulate dosage unit forms for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to the physical unit of dosage set as a single dose for the subject to be administered. Each unit contains a predetermined amount of the active compound calculated to obtain the desired therapeutic effect in association with the desired pharmaceutical carrier. The instructions for the dosage unit form of the present invention are dictated by and dependent upon the inherent characteristics of the active compound and the particular therapeutic effect to be achieved, and the inherent limitations of the art of constructing the active compound for individual treatment.

The pharmaceutical composition may be included in a container or pack or dispenser with instructions for administration.

Diagnostic method

The huPD-L1 antibody of the present invention provides a method of detecting a tissue that is susceptible to cancer by being easily "cancerous" or abnormal cell proliferation when bound to a detectable moiety. In addition to tissue that becomes cancerous due to in situ neoplasia, for example, an antibody detectable partial conjugate also provides a method for detecting cancer metastatic tissue present in end organs and / or tissues. Thus, the tissue can be detected by contacting the suspected cancerous tissue with an antibody detectable moiety under suitable conditions such that the detectable moiety can be detected in the cancerous tissue, detecting the presence of cancerous tissue.

The huPD-L1 antibody of the present invention provides a method for detecting T cell depletion in a subject suffering from cancer or chronic infection when bound to a detectable moiety. For example, the huPD-L1 antibody may be used to detect the level of PD-L1 in a subject, and may be compared to a baseline level to indicate whether the subject is experiencing T cell depletion. Thus, the method can also be used to determine whether treatment with the huPD-L1 antibody that enhances the immune response by reversing or inhibiting T cell depletion is beneficial to the subject.

The detectable moiety can be conjugated directly or indirectly to the antibody or fragment using, for example, a fluorescent secondary antibody. Direct conjugation can be performed, for example, by standard chemical bonding of the fluorescent moiety to the antibody or antibody fragment, or through genetic engineering. The chimeric or fusion protein can be engineered to include an antibody or antibody fragment that is attached to a fluorescent or bioluminescent protein. For example, Casadei et al. Discloses a method for producing a vector construct capable of expressing a fusion protein of an ecorin and an antibody gene in mammalian cells.

The term " labeled " in the context of a probe or antibody refers to the ability of a detectable substance to bind (i.e., physically bind) a probe or an antibody to directly label the probe or antibody, and by reactivity with another indirectly labeled reagent Quot; means to indirectly label the probe or antibody. Examples of indirect labeling include detection of a primary antibody that can be detected as fluorescently labeled streptavidin using a fluorescently labeled secondary antibody and a biotin with a DNA labeling end-label. The term " biological sample " refers to tissues, cells and biological fluids isolated from a subject (e.g., a biopsy) and tissues, cells, and body fluids present in the subject. That is, the detection method of the present invention can be used to detect cancer, cancer cells or cancer-associated cells (for example, a tumor or an interstitial cell associated with cancer cells) in an in vitro and in-vivo biological sample. For example, in vitro techniques for PD-L1 detection include enzyme linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. In vivo techniques for the detection of PD-L1 also include the step of introducing the labeled anti-PD-L1 antibody into the subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques. In an embodiment, the invention provides a non-invasive method of detecting tumor or cancer cells in a subject. The subject is administered an antibody or scFv antibody of the invention and the antibody can be conjugated to a detectable moiety (i. E., A fluorescent, chemical, chemiluminescent, radioactive, or any other detectable moiety Portion), and the antibody is detected by observation of the detectable moiety after allowing it to localize in the tumor.

In the case of a " targeted " conjugate, i.e. a targeting moiety-a conjugate comprising a molecule or feature designed to localize the conjugate at a particular site or sites within the animal or animal, the localization may be "localized" Represents the state when the equilibrium has necessarily been reached between the free, " liberated " The rate at which the equilibrium is reached is determined by the route of administration. For example, conjugates administered by intravenous injection to localize in the thrombus can be placed or accumulated in the thrombus within minutes after injection. On the other hand, the conjugate administered orally to localize to an intestinal infection may take several hours to be deployed. Alternatively, the localization may simply indicate the localization of the entity in the subject or animal at the selected time after the individual is administered. As another example, localization occurs when a portion is dispensed after administration.

In all of the above cases, the appropriate estimated time at which the localization is achieved may be determined by the person skilled in the art. In addition, the localized state as a function of time can be traced by imaging a detectable moiety (e. G., A light emitting conjugate) according to the method of the present invention, for example, using an optical detection device. The " light detection device " used must have a sensitivity high enough to enable the imaging of faint light from within the mammal within a reasonable amount of time and to utilize the signal from the imaging device.

In cases where too bright a light generating moiety can be used and / or the localized light generating fusion protein can be detected near the surface of the subject or animal to be imaged, a "night-vision" goggle or silicone intensifier tube (SIT) camera (e.g., purchased from Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. More typically, however, a more sensitive optical detection method is required.

If the luminosity is too low, the photon flux per unit area is too low to make the scene to be the image no longer continuous. Instead, they appear as individual photons that are temporally spatially different. When shown on the monitor, the image appears as a scintillation point of light, each representing a single detected photon. Over time, the photons detected in the digital image processor can be accumulated to obtain and produce an image. Compared to a conventional camera that assigns a signal at each image point to an intensity value, the amplitude of the signal is not significant in photonic counting imaging. The goal is to simply detect the presence of a signal (photon) and count the occurrence of a signal over its position over time.

Two or more types of optical detection devices described below can detect individual photons and generate signals that can be analyzed by an image processor. Noise reduction photodetectors reduce the fundamental noise in the photon detector while amplifying the photon signal to obtain sensitivity. Noise is mainly reduced by cooling the detector array. The device includes a charge coupled device (CCD) camera called a " backthinned " cooled CCD camera. In more sensitive devices, cooling is accomplished, for example, using liquid nitrogen to bring the temperature of the CCD array to approximately -120 ° C. &Quot; Backscin " refers to an ultra-thin back plate that increases the quantum efficiency by reducing the path length that photons follow to be detected. A particularly sensitive backscin cryogenic CCD camera is the series 200 camera "TECH512", available from Photometrics, Ltd. (Tucson, Ariz.).

A " photon amplifying device " amplifies a photon before the photon strikes the detection screen. This type is a camera with an amplifier such as a microchannel amplifier. Microchannel amplifiers typically include a channel of a metal array that is perpendicular or has the same width as the detection screen of the camera. The microchannel array is placed between the sample, the subject, or the animal and the camera that will be the image. Most of the photons entering the channel of the array touch the sides of the channel before emerging. The voltage applied through the array causes a lot of electrons to be emitted from each photon impingement. The electrons from the collision leave their original channel in a " shotgun " pattern and are detected by the camera.

Much greater sensitivity can be achieved by placing the amplified microchannel array in the series to produce the electrons generated in the first stage in turn as electrons in the amplified signal in the second stage. However, the sensitivity increases with the cost of spatial resolution, which reduces each additional step of amplification, a typical microchannel amplifier based single photon detection device is the C2400 series available from Hamamatsu.

 An image processor processes signals generated by a photodetector device that counts photons, for example, to produce images that can be viewed on a monitor or printed on a video printer. The image processor is typically sold as part of a system that includes the sensitive photon counting camera described above or is available from the same source. The image processor is typically connected to a personal computer such as an IBM-compatible PC or Apple Macintosh (Apple Computer, Cupertino, Calif.), Which may or may not be included as part of the purchased imaging system. Once the image is in digital form, it can be manipulated and printed by various image processing programs (e.g., " ADOBE PHOTOSHOP ", Adobe Systems, Adobe Systems, Mt. View, Calif.).

In one embodiment, the biological sample comprises protein molecules of a test subject. One of the preferred biological samples is a peripheral blood lymphocyte sample isolated from the subject by conventional methods.

The present invention also includes a kit for detecting the presence of PD-L1 or PD-L1-expressing cells in a biological sample. For example, a kit can determine the amount of PD-L1 in a sample, a labeled compound or agent (e.g., anti-PD-L1 scFv or monoclonal antibody) capable of detecting cancer or tumor cells in a biological sample And means for comparing the amount of PD-L1 in the sample with a standard. The standard is, in some embodiments, a non-cancer cell or an extract thereof. The compound or preparation may be packaged in a suitable container. The kit may also include instructions for using the kit to detect the cancer in the sample.

Bispecific antibody

A bispecific antibody (bsAb) is an antibody comprising two variable domains or scFv units, and the resulting antibody recognizes two different antigens. The present invention provides bispecific antibodies that recognize PD-L1 and a second antigen. Typical second antigens are tumor-associated antigens, cytokines, and cell surface receptors. In some embodiments, the second antigen may be CAIX (carbonic anhydrase IX, or G250), IL-10 or CCR4. In some embodiments, the second antigen may be a cell surface receptor, wherein the cell surface receptor is CCR4, IL21R, BTLA, HVEM or TIM3.

Bispecific antibodies of the invention include heavy chain and light chain combinations or scFv of the huPD-L1 antibodies disclosed herein.

Production of bispecific antibodies

The bispecific antibodies of the present invention can be produced using methods known in the art. In some embodiments, bispecific antibodies are single polypeptides in which two scFv units are linked by a long linker polypeptide of sufficient length to allow for an intramolecular association between the two scFv units forming the antibody. In another embodiment, bispecific antibodies are one or more polypeptides that are joined by covalent or noncovalent bonds.

In another embodiment, bispecific antibodies are made using the " handle to the hole " method (Ridgway et al., Protein Eng 7: 617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively destroy heavy chain pairing while maintaining heavy-light chain pairing. The two heavy chain-light chain heterodimers that recognize two different antigens are mixed to promote heterotypic pairing mediated through the manipulated " handle to hole " of the CH3 domain as shown in Figures 5 and 6A.

In another embodiment, the bispecific antibody comprises a heavy chain-light chain duplex from two or more different antibodies so that the first heavy chain-light chain duplex recognizes PD-L1 and the second heavy chain-light chain duplex forms a hybrid antibody recognizing the second antigen. Can be produced through the exchange of a light chain dimer. The mechanism for heavy chain-light chain duplexes is similar to the formation of human IG4, which also functions as a bispecific molecule. The coupling reaction of the IgG heavy chain is induced by an intramolecular force, for example, pairing of the CH3 domain of each heavy chain with a disulfide bridge. The presence of a particular amino acid (R409) within the domain was shown to facilitate dimer exchange and production of IgG4 molecules. The heavy chain pairing is also stabilized by disulfide bridging between the heavy chains in the hinge region of the antibody. In particular, in IgG4, the hinge region comprises the amino acid sequence Cys-Pro-Ser-Cys (compared to the stable G1 hinge region comprising the sequence Cys-Pro-Pro-Cys) at amino acids 226-230. This difference in sequence of serine at position 229 is associated with a tendency for IgG4 to form a new intrachain disulfide bond within the hinge region (Van der Neut Kolfschoten, M. et al., 2007, Science 317: 1554-1557) And (Labrijn, AF et al, 2011, Journal of immunol 187: 3238-3246).

Thus, the bispecific antibodies of the invention can be generated by introduction of a Cys-Pro-Ser-Cys sequence in the hinge region of an antibody that recognizes the R409 residue and the PD-L1 or the second antigen in the CH3 domain, Wherein said heavy chain-light chain duplex recognizes a heavy chain-light chain duplex as long as it recognizes PD-L1 and a second heavy chain-light chain duplex that recognizes a second antigen, said second antigen being selected from any of the antigens to be. Preferably, the bispecific antibody comprises an anti-PD-L1 heavy chain-light chain duplex conjugated to the anti-CAAX (carbonic anhydrase IX, or 250) heavy chain-light chain duplex as discussed in Examples 5 and 6 do. The heavy chain-light chain dimer exchange can also be elevated by the addition of a reducing agent that promotes exchange, for example, reduced glutathione. The known IgG4 molecules may also be modified such that the heavy and light chains recognize PD-L1 or the second antigen as disclosed herein. The use of this method of producing the bispecific antibodies of the present invention is advantageous in that the Fc region does not interact with other IgG subtypes in that it does not interact with the effector system of the immune response, Lt; RTI ID = 0.0 > IgG4 < / RTI > Due to these unique properties, the IgG4-based bispecific antibodies become attractive for therapeutic applications where antibodies bind to the target and functionally alter the target and associated signaling pathways but do not induce effector activity.

In some embodiments, the mutation is introduced into the constant region of bsAb to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of bsAb. The above embodiment is illustrated in Fig. 6B. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the bsAb comprises a mutation on one scFv unit of the heterodimer bsAb, which reduces ADCC activity. In another aspect, bsAb comprises a mutation in both chains of the heterodimeric bsAb, which completely eliminates ADCC activity. For example, a mutation introduced into one or both of the scFv units of bsAb is a LALA mutation of the CH2 domain. The bsAb with variable ADCC activity exhibits the greatest selective killing for cells expressing an antigen recognized by bsAb but can be optimized to exhibit minimal killing for a second antigen recognized by bsAb have.

A typical second antigen

The present invention RTI ID = 0.0 > PD-L1 < / RTI > and a second antigen.

In some embodiments, the second antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is carbonic anhydrase IX (CAIX). For example, a CAIX / PD-L1 bispecific antibody can be made to comprise a combination of one light chain with one heavy chain of the huPD-L1 antibody described herein and one light chain and one light chain that recognize CAIX (Figure 5A ). CAIX has been described as a target of immunotherapy using IL-2 and prognostic markers of disease progression. CAIX is a tumor-associated antigen that is highly expressed in cancers such as renal cell carcinoma. In some cases, activation of the PD1 / PD-L1 axis by tumor cells can induce T cell depletion against certain tumor-specific antigens such as CAIX, thereby allowing tumor cells expressing CAIX to avoid recognition by the immune system do. The bsAb targeting both CAIX and PD-L1 serves as a new cancer treatment. Treatment with CAIX / PD-L1 bsAb inhibits or reverses PD-1 / PD-L1-mediated T cell depletion to CAIX and promotes tumor surveillance and immune response to CAIX-expressing tumor cells will be. For example, the targeted antigen will promote an antigen-specific immune response to tumor cells that are CAIX.

In some cases, the mutation may be introduced into the constant region (e. G., The CH2 domain) in the CAIX or PD-L1 chain to reduce ADCC activity (Figure 5B). In some cases, the mutation may be introduced into a constant region (e. G., The CH2 domain) in the CAIX or PD-L1 chain to eliminate ADCC activity. Mutated bsAbs with variable ADCC activity can be analyzed by methods known in the art for the specificity of killing tumor cells. Preferably, the CAIX / PD-L1 bsAb will exhibit maximum selective killing of CAIX-expressing tumor cells, but exhibit minimal killing of PD-L1-expressing endogenous peripheral blood mononuclear cells (PBMC).

In some embodiments, the second antigen is interleukin 21 (IL21R) as a cell surface receptor. For example, an IL21R / PD-L1 bispecific antibody can be made to comprise a combination of one light chain with one heavy chain of the huPD-L1 antibody described herein and one light chain and one light chain that binds agonistically to the IL21R have. Cytokine IL21 is secreted by CD4 + T helper cells and binds to IL21R promoting some immunological activation pathways, particularly promoting antigen-specific cytotoxicity of CTLs and maturation, proliferation and cytotoxicity of NK cells (Sondergaard , H et al., 2009 Tissue antigens 74: 467-479) (Kasaian, MT et al, 2002 Immunity 16: 559-569); And (Coquet, JM et al, 2008 J Immunol 178: 2827-2834). In particular, it has been shown that IL21 promotes melanoma antigen activation and cytotoxic performance thereto (Li, Y. et al, 2005, J Immunol 175: 2261-2269). Systemic administration of IL21 has been studied for cancer treatment applications and has shown some efficacy (Schmidt, H. et al, 2010 Clinical cancer research , 16: 5312-5319), some data suggest that harmful and undesirable (Grunwald, V. et al, 2011, Acta Oncol 50: 121-126).

The IL21R / PD-L1 bsAb of the present invention binds IL21R in an agonist manner and acts as a mimic or surrogate of cytokine IL21. Binding by IL21R / PD-L1 bsAb results in the activation of the IL21R-mediated pathway and the promotion of antigen-specific cytotoxic immune responses to tumor cells leading to subsequent PD-L1-mediated T cell depletion. A particular advantage of treatment with the bispecific antibodies of the invention is the ability to induce T cell depletion to avoid PD-1 / PD-L1-mediated T cell depletion, e.g., anti-tumor immune response Localize < RTI ID = 0.0 > IL21R < / RTI > activation in the microenvironment of the tumor, e.g., near the tumor or in the tumor. In this way, the IL21R / PD-L1 bsAb has been shown to inhibit or inhibit 1) the PD-1 / PD-L1-mediated T cell depletion and 2) the IL21 / IL21R- Promotes tumor-mediated activation by inducing antigen-specific or anti-tumor immune responses and cytotoxicity by promoting mediated activation.

In addition, IL21R / PD-L1 bispecific antibodies may have utility as vaccines or vaccine adjuvants for vaccination of subjects. (GCR) in which high affinity antibody-secreting plasma cells and memory B cells are produced that ensure sustained immunoprotection and a rapid recall response to previously encountered antigens, PDL1 is expressed in embryonic B cells PD1 is expressed in follicular T-helper cells (TFH) (Crotty, S. et al, 2011, Annual review of Immunology , 29: 621-623). Overexpression of PD1 or PD-L1 inhibits the expansion of antibody-producing B cells. Use of the IL21R / PD-L1 bispecific antibody, in which the PD-L1 portion of the antibody inhibits the PD1 / PD-L1 axis, will preferentially expand only the high affinity antibody. Since IL-21 strongly promotes the transfer of antigen-specific B cells to antibody-secreted plasma cells and the formation and persistence of TFH activity (Crotty, S. et al, 2011, Annual review of Immunology , 29 : 621-623)), bsA for IL21 surrogate or PDLl with agonist activity is specific for the stimulation of high affinity antibody production for an antigen that is administered via a particular antigen, for example, a vaccine, or an antigen of the infectious agent It can act as an enantiomer.

In some embodiments, the second antigen is BTLA (B and T lymphocyte attenuator protein) or HVEM (herpes virus entry mediator, also known as TNFRSF14). For example, a BTLA / PD-L1 bispecific antibody can be made to comprise a combination of one light chain with one heavy chain of the huPD-L1 antibody described herein and one light chain and one light chain that binds to BTLA. For example, an HVEM / PD-L1 bispecific antibody may be conjugated to a heavy chain and a light chain combination of the huPD-L1 antibody described herein, and to a HVEM, preferably a heavy chain that binds to the region of the HVEM that mediates binding with BTLA And one light chain. BTLA binding to HVEM results in T cell inhibition similar to the PD1 / PD-L1 interaction. The BTLA / PD-L1 or HVEM / PD-L1 bispecific antibodies of the invention inhibit or prevent association between BTLA and HVEM and prevent BTLA / HVEM-mediated T cell inhibition. BTLA inhibition promotes tumor-specific T cell responses. Thus, treatment with the bispecific antibodies of the present invention simultaneously blocks two different inhibitory pathways that limit T cell activity.

In some cases, the HVEM / PD-L1 bsAb antibody will also prevent association between HVEM and another HVEM ligand, CD160. CD160 is an agonist for the survival of B cell chronic lymphocytic leukemia (BCLL) cells and has been shown to strongly inhibit CD4 + T cell activation and function. Treatment with the HVEM / PD-L1 bispecific antibody, which also prevents the HVEM / CD160 binding of the present invention, simultaneously blocks two inhibitory pathways that limit T cell activity and inhibition of the CD160-mediated tumor survival pathway.

In some embodiments, the second antigen is TIM3 (T cell immunoglobulin and mucin domain 3). For example, a TIM3 / PD-L1 bispecific antibody can be made to comprise a light chain combination with one heavy chain of the huPD-L1 antibody described herein, and a combination of one light chain and one light chain that binds to TIM3. In one aspect, the antibodies of the invention prevent or inhibit galectin-9 (GAL9) and TIM3 binding. The interaction between TIM3 and GAL9 results in inhibition of T cell function and activation of macrophages (Sakuishi, K. et al, 2010, The Journal of experimental medicine, 207: 2187-2194) and (Zhang, Y. et al. et al., 2012, Journal of leukocyte biology, 91: 189-196))). TIM3 also has been shown to promote the activity of bone marrow-derived inhibitory cells (MDSC) (Dardalhon, V. et al., 2012 Journal of leukocyte biology , 185: 1383-1392). Thus, treatment with the bispecific antibodies of the invention, such as TIM3 / PD-L1 bsAb, will reverse and prevent T cell depletion, promote tumor monitoring and inhibit the production of MDSC. The use of TIM3 / PD-L1 bsAb may be particularly advantageous for the treatment of subjects with TIM3 polymorphisms, such as renal cell carcinoma and metastatic renal cell carcinoma.

Bispecific antibodies disclosed herein may be useful in the treatment of a disease or disorder, such as cancer. In particular, the bispecific antibodies of the invention may be useful in diseases or disorders associated with T cell depletion. In some cases, the bispecific antibodies disclosed herein may be used as a vaccine to promote an antigen-specific immune response. The bispecific antibodies of the invention will target tumors that induce T cell depletion.

Treatment method

The present invention provides both prophylactic and therapeutic methods for treating subjects at risk for (or susceptible to) cancer or other cell proliferation related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with, for example, abnormal expression of PD-L1. For example, the method can be used to treat, prevent or alleviate symptoms of renal cell carcinoma or breast cancer. Alternatively, the method can be used to treat, prevent, or alleviate symptoms of cancer in which PD-L1 plays a negative regulatory role in T cell responses. Alternatively, the method can be used to treat, prevent, or alleviate symptoms of solid tumors, such as breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, or stomach cancer . Alternatively, the method can be used to treat, prevent, or alleviate the symptoms of metastatic cancer

The present invention provides both prophylactic and therapeutic methods of treating subjects at risk of (susceptible to) chronic viruses, bacterial or parasitic infections. In particular, the present invention provides both prophylactic and therapeutic methods of treating subjects at risk of (susceptible to) HIV infection or AIDS.

The present invention also provides both prophylactic and therapeutic methods for treating subjects at risk of developing a T cell depletion or at risk of a disease or disorder or condition associated with T cell depletion. Such diseases or disorders include, but are not limited to, HIV, AIDS, and chronic bacterial, viral or parasitic infections. Other chronic infections include, for example, hepatitis B virus (HBV), infectious C virus (HCV), herpes simplex virus 1 (HSV-1), H. Pylori (H. pylori), or Toxoplasma Oregon DI (Toxoplasma gondii ) .

Accordingly, in one aspect, the invention provides a method of preventing, treating, or alleviating a symptom of a cancerous or cellular proliferative disorder or disorder in a subject by administering to the subject a monoclonal antibody or scFv antibody of the invention. For example, the huPD-L1 antibody can be administered in a therapeutically effective amount.

Subjects at risk for a cancer or cell proliferation related disease or disorder include subjects having a family history of cancer or subjects exposed to known or suspected cancer inducers. By administering a prophylactic agent before the onset of cancer, the disease can be prevented or, alternatively, delayed in its progress.

In another aspect, contact with a PD-L1 antibody of the invention inhibits tumor cell growth or reduces inhibitory T cell activity. Cells are any cells that express PD-L1. For example, the cell is a T cell.

The invention also encompasses methods of increasing or augmenting an immune response to an antigen. An immune response is increased or increased by administering the monoclonal or scFv antibody of the invention to the subject. The immune response is increased, for example, by enhancing antigen-specific T effector function. The antigen is a virus (e. G., HIV), a bacterium, a parasite, or a tumor antigen. The immune response is a natural immune response. A natural immune response is an immune response that is the result of an infection. Infection is a chronic infection. An increase or increase in the immune response to an antigen can be measured by a number of methods known in the art. For example, the immune response can be measured by measuring any of the following: T cell activity, T cell proliferation, T cell activation, production of effector cytokines, and T cell transcription profile.

Alternatively, the immune response is a response induced by the vaccination. Thus, in another aspect, the invention provides a method of increasing the efficiency of a vaccine by administering a monoclonal antibody or scFv antibody and vaccine of the invention to a subject. Antibodies and vaccines may be administered sequentially or concurrently. The vaccine is a tumor vaccine, a bacterial vaccine or a viral vaccine.

How to combine

The present invention provides for the treatment of cancer in a patient by administering two antibodies that bind to two identical epitopes of the PD-L1 protein or, alternatively, two different epitopes of the PD-L1 protein. Alternatively, the cancer is treated by administering a first antibody that binds to PD-L1 and a second antibody that binds to the protein in addition to PD-L1. For example, other proteins besides PD-L1 may include, but are not limited to, CAIX, CCR4, and IL-10. For example, other proteins besides PD-L1 are tumor-associated antigens.

In some embodiments, the invention provides huPD-L1 antibody administration alone or in combination with cells capable of causing or augmenting an immune response with additional antibodies that recognize another protein other than PD-L1. For example, the cells may be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMC, such as cytotoxic T cells, macrophages, and natural killer cells (NK) cells.

The present invention also provides antibodies and anti-neoplastic agents that bind to the PD-L1 protein, such as small molecules, growth factors, cytokines or peptides, peptide mimetics, peptoids, polynucleotides, lipid- And other therapeutic agents including biomolecules such as primary amines, hormones, neuropeptides and proteases. Small molecules include, but are not limited to, inorganic molecules and organic small molecules. Suitable growth factors or cytokines include IL-2, GM-CSF, IL-12, and TNF-alpha. Small molecule libraries are known in the art (see Lam, Anticancer Drug Des., 12: 145,1997)

The present invention will also be described in the following examples, which do not limit the scope of the invention described in the claims.

Example

Example  1: PD- L1 Human mAb Creation of

A human scFv phage display library of 27 billion members was selected to generate mAbs for human PD-L1. It was confirmed that 14 unique scFv-phages bind to PD-L1 using full length PD-L1 in the form of superparamagnetic protein liposome (PMPL), which ensures the proper orientation of the PD-L1 extracellular domain for presentation in the library . Human IgG constructs were prepared from the fourteen unique scFv-phage: Ab-14, Ab-16, Ab-22, Ab-30, Ab-31, Ab-32, Ab-38, Ab-42, -50, Ab-52, Ab-55, Ab-56 and Ab-65.

Example  2: PD- L1 For huPD - L1 mAb  Investigate the nature of bonding

Binding analysis of huPD-L1 antibody was carried out using PD-L1-expressing cells. Including the parental cell line 300.19, and the transfected cell line expressing human PD-L1 (hPD-L1), human PD-L2 (HPD-L2), and human C-type lectin domain family 2 member (hCLEC2D) Cells were tested. Binding analysis was repeated twice, and the results are summarized in Tables 16-19 and 2 below. The antibody affinity was tested using four antibody concentrations of 10 / / ㎖, 1 / / ㎖, 0.1 / / ㎖ and 0.01 / / ㎖. GF1538 and GF1757 were used as controls. GF1538 is a humanized Ab for hPD-L1 and GF1757 is a humanized Ab for hPD-L2. The secondary antibody used was PE-goat anti-human IgG. All values are expressed as mean fluorescence intensity (GMFI) detected by FACS analysis.

Binding assay results indicate that the tested huPD-L1 antibody shows high affinity and specificity for binding PD-L1. A parental cell line 300.19 that did not express human PD-L1 as a control was used to confirm basal or nonspecific fluorescence (Table 16 and [Figure 2] upper left). The staining of 300.19 cells expressing human PD-L1 by transfection with the huPD-L1 antibody showed significantly higher mean fluorescence intensity (MFI), demonstrating that the antibody binds to PD-L1. Even at the lowest dilution of 0.01 [mu] g / ml antibody, a significant MFI value demonstrated a high affinity of the huPD-L1 antibody for PD-L1 protein binding (Tables 17A, 17B and [Fig. As demonstrated in Tables 18A, 18B, 19A, 19B and [Figure 2] (lower two graphs), the huPD-L1 antibody demonstrated little binding to human PD-L2 or CLEC2D when expressed in 300.19 cells . However, the MFI values obtained in the staining of PD-L2 and CLE2D were not as high as those obtained in PD-L1. Also, at lower dilution rates, the MFI value was not significantly higher than the baseline level, indicating that the huPD-L1 antibody does not have high affinity or specificity for PD-L2 or CLE2D. Some huPD-L1 antibodies, such as Ab-42, demonstrated some cross-reactivity with PD-L2, but the antibodies have significantly higher affinity and specificity for PD-L1.

Example  3: PD / PD- L1  The anti-PD- L1 Phage - Characterization of antibodies

FACS competition analysis was performed to investigate the properties of inhibition of hPDl binding to hPD-L1 by anti-PD-L1 phage antibody. All anti-hPD-L1 antibodies were in phage-scFv form. 293T cells were transfected with a vector encoding human PD-L1 fused to the human Fc region for hPD-L1-Fc expression. In this assay, phage-scFv of 10 ¹² plaque forming units (pfu) was added to hPD-L1-expressing 293T cells after mixing with approximately 0.25 mg / ml of soluble hPD-1-hFc fusion protein. After washing, cells were incubated with FITC-anti-human IgG antibody and analyzed by FACS to measure hPDl-hFc binding to cellular hPD-L1.

Fluorescence values were obtained by FACS analysis and used to obtain inhibition rate of hPD-1 binding to hPD-L1 + cells. The above values are shown in Fig. Almost all of the anti-PD-L1 phage scFvs were able to suppress hPD-1 binding to hPD-1. In particular, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52 and Ab-55 phage-scFv significantly increased hPD-1 / hPD- .

Example  4: PD / PD- L1  To block binding huPD - L1  Availability mAb Characterization of

FACS competition analysis was performed to characterize the inhibition of hPDl binding to hPD-L1 by the soluble huPD-L1 antibody of the present invention. All huPD-L1 antibodies were tested for their ability to inhibit the binding of hPD-L1-expressing 300.19 cells to the hPD1-IgG fusion protein. In this assay, 50,000 cells expressing hPD-L1 were pre-incubated with huPD-L1 or control antibody for 30 minutes at the following concentrations: 10 μg / ml, 1 μg / ml, 0.1 μg / ml and 0.01 μg / ml. After incubation, human PD1 fused to 0.125 [mu] g of mouse IgG2 [alpha] was added to the cells and incubated for an additional 30 minutes. After washing the cells twice, 0.125 [mu] g of goat anti-mouse IgG2 [alpha] -PE antibody was added to the cells. After incubation for 30 minutes, the cells were washed twice and analyzed by FACS. The values obtained in the FACS are expressed in mean fluorescence intensity units (MFI) and summarized in Table 20. Using the MFI values in Table 20, the inhibition rate of hPDl binding to hPD-L1 + cells of [Fig. 4] was obtained. As demonstrated herein, all of the huPD-L1 soluble antibodies tested demonstrated almost complete inhibition of hPD-L1 and hPDl binding at 10 [mu] g / ml. At successively lower concentrations, most soluble antibodies, particularly Ab-42 and Ab-50, still demonstrate very good inhibition of PDl / PD-L1 binding.

Example  5: Formation of bispecific antibodies

Based on the role of the hinge in generating half units of IgG ₄ molecules, the introduction of charged mutations in the hinge region of human IgG ₁ not only facilitates half-monomer exchange, It is possible to stabilize it. This example demonstrates that the combination of hinge and CH3 mutation increases bispecific antibody formation.

In order to further stabilize heterodimer formation, the opposite charged mutation was also replaced by the CH3 domain, which is also a " handle to hole " concept. A bispecific antibody can be formed through the following steps. First, two parental antibodies containing a bispecific mutation were expressed and purified, respectively. The antibody is then mixed in the presence of a weak reducing agent. The weak reduction of the antibody resulted in dissociation into two monomers each having a variable heavy chain and a variable light chain. Subsequently, the monomers were mixed with each other and then subjected to an oxidation step to cause the formation of bispecific antibody molecules.

PD-L1 and G250 (carbonic anhydrase IX). Anti-G250 parent (G37 wild type, G37WT) and engineered (G37 KIHA) antibodies were generated and purified into two independent vectors. G37 KIHA, which incorporates a double-specific mutation following the "handle to hole" concept, has been altered in the immunoglobulin hinge region.

To understand the dissociation activity of the G37 KIHA, the antibody was mixed in the presence of a weak reducing agent to inhibit the formation of antibody monomers by different concentrations of glutathione (GSH) (Fig. 7A). As shown by increasing levels of low molecular weight species in [Figure 7A], the addition of reducing agent (GSH) at increasing concentrations led to dissociation of the antibody into monomers.

Similarly, anti-PD-L1 parents (PD-L1 wild type, PDL-1WT) and engineered (PD-L1 KIHB) antibodies were generated and purified into two independent vectors. Various concentrations of GSH were used to identify suitable conditions for obtaining anti-PD-L1 monomers (Fig. 7B). The best conditions of GSH concentration were selected, and the resulting anti-PD-L1 monomers were incubated with G37 KIHA monomers. It has been shown that bispecific antibodies are formed in the same size as wild-type IgG, indicating that two heavy chain-light chain units, one PD-L1-specific monomer and the other has generated an antibody comprising a G250-specific monomer .

Example  6: Bispecific antibody function

The bispecific antibodies generated in Example 5 were then tested for their ability to recognize both antigens, e.g., PD-L1 and G250. The function of PD-L1 and G250 bispecific antibodies was tested using flow cytometry. CAIX ⁺ PD-L1 ^- SKRC-52 expressed CAIX (250) but not PD-L1. Thus, cells could only be recognized by anti-CAIX G37 and could not be recognized by PD-L1. To avoid saturation of antibody binding, antibody concentrations were made low and reduced dosing. Indeed, parental anti-G37 recognized SKRC-52 cells, while parental anti-PD-L1 was not recognized (the same level as the control). Bispecific antibodies comprising conjugated anti-G37 and anti-PD-L1 monomers recognized SKRC-52 cells at half-reduced concentrations as compared to the parental G37 antibody, thereby using the methods and antibodies described herein The functionality of the resulting bispecific antibody was demonstrated.

Example  7: mAb42 Functional Characteristics

In addition, the functional characteristics of the mAb42 to PD-L1 were examined. Peripheral blood mononuclear cells (PBMC) of four different healthy donors (D1-D4) were cultured. PBMC were cultured in the presence of mAb42 or in the presence of control allogeneic IgG antibody. PMBC was stimulated with 0.1 μg / ml SEB (Endotoxin B of Staphylococcus aureus) and TNFα production was measured using MSD (Meso Scale Delivery). Sample analysis was repeated three times.

When cultured in the presence of anti-PD-L1 antibody (mAb42) compared to the control antibody in all four donor samples as shown in Figure 8, PBMC cultures in the presence of mAb42 increased TNF [alpha] production in response to SEB . In addition, the increase in TNFa production was statistically significant at p <0.0005. Thus, treatment with an anti-PD-L1 antibody increases the immune response in response to an antigen or infection in humans.

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

                         SEQUENCE LISTING <110> Dana-Farber Cancer Institute, Inc.        Marasco, Wayne A.        Sui, Jianhua   <120> Human Monoclonal Anti-PD-L1 Antibodies and Methods of Use <130> 20363-068001WO <140> PCT / US13 / 63509 <141> 2013-10-04 &Lt; 150 > US 61 / 709,731 <151> 2012-10-04 &Lt; 150 > US 61 / 779,969 <151> 2013-03-13 <160> 140 <170> PatentIn version 3.5 <210> 1 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-14 <400> 1 caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagctcta 300 cctagtggga ctatactggt cggaggttgg ttcgacccct ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210> 2 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-14 <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu     50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ala Leu Pro Ser Gly Thr Ile Leu Val Gly Gly Trp Phe Asp             100 105 110 Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 3 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-14 <400> 3 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg caacattgcc agcaattatg tgcagtggta ccaacagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcag caatctttgg 300 gtgttcggcg gagggaccaa gctgaccgtc cta 333 <210> 4 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-14 <400> 4 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Asn Ile Ala Ser Asn             20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val         35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser     50 55 60 Gly Ser Ile Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser                 85 90 95 Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 5 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-16 <400> 5 gaggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgccc tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggtg gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagacgtg 300 tttccagaga ctttttcgat gaactacggt atggacgtct ggggccaagg aaccctggtc 360 accgtctcct ca 372 <210> 6 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain Ab-16 <400> 6 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Ser Gly Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Asp Val Phe Pro Glu Thr Phe Ser Met Asn Tyr Gly Met Asp             100 105 110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 7 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-16 <400> 7 tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60 acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca gaagccagga 120 caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180 ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240 gatgaggctg actattactg taactcccgg gacagcagtg gtaaccatta tgtcttcgga 300 actgggacca aggtcaccgt ccta 324 <210> 8 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-16 <400> 8 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala             20 25 30 Ser Trp Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr         35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser     50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His                 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu             100 105 <210> 9 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-22 <400> 9 caggtgcagc tggtgcagtc tgggggaggc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccgtcaagct 120 ccagggaagg gtctggagtg ggtctctctt attagtgggg atggtggtag cacatactat 180 gcagactctg tgaagggccg attcaccatc tccagagaca acagcaaaaa ctccctgtat 240 acgtaggag ctcccctgta gtagtaccag ctgctatgga agcgtcggtg cttttgatat ctggggccaa 360 gggaccacgg tcaccgtctc ctca 384 <210> 10 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-22 <400> 10 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Leu Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Lys Val Leu Leu Pro Cys Ser Ser Thr Ser Cys Tyr Gly Ser Val             100 105 110 Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 125 <210> 11 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain Ab-22 <400> 11 taggacgatg agctcggtcc cagctccgaa gacataatga tcactattat tatcccacac 60 ctgacagtaa taatcggcct catcaccggc ttcgaccctg ctgatggtca gggtggccgt 120 gttcccagag ttggagccag agaatcgctc agagatccct gagggccggt ccctatcaga 180 gtagatgacc aacgcagggg cctggcctgg cttctgctgg taccagtgca cactcttcct 240 tccaatgtcg cttcccccac aggtaatcct ggccgtcttt cctggggcca ctgacactga 300 gggtgcctga gtcagcacag gcag 324 <210> 12 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-22 <400> 12 Leu Pro Val Leu Thr Gln Ala Pro Ser Val Ser Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Ser Asp Ile Gly Arg Lys Ser Val             20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ala Leu Val Ile Tyr         35 40 45 Ser Asp Arg Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser     50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Asn Ser Asp His                 85 90 95 Tyr Val Phe Gly Ala Gly Thr Glu Leu Ile Val Leu             100 105 <210> 13 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-30 <400> 13 caggtgcagc tggtgcagtc tgggggaagt gtggtacggc ctggggaatc cctcagactc 60 tcctgtgtag cctctggatt catctttgat aattatgaca tgagttgggt ccgccaagtt 120 ccagggaagg ggctggagtg ggtctctcgt gttaattgga atggtggtag cacaacttat 180 gcagacgctg tgaagggccg attcaccatc tccagagaca acaccaagaa ctccctgtat 240 acacgatag gtcggtgctt atgatctctg gggccagggg accacggtca ccgtctcctc a 351 <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-30 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Gly Ser Val Val Arg Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Asp Asn Tyr             20 25 30 Asp Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Arg Val Asn Trp Asn Gly Gly Ser Thr Thr Tyr Ala Asp Ala Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Arg Glu Phe Val Gly Ala Tyr Asp Leu Trp Gly Gln Gly Thr Thr             100 105 110 Val Thr Val Ser Ser         115 <210> 15 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-30 <400> 15 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacaa 120 cacccaggca aagcccccaa actcatgatt tatgatgtca gtaatcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatata caagcagcac tctgccgttc 300 ggcggaggga ccaagctgac cgtccta 327 <210> 16 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-30 <400> 16 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr             20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu         35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe     50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser                 85 90 95 Thr Leu Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 <210> 17 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-31 <400> 17 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc caggggccac agtgaaggtc 60 tcctgcaagg tttttggaga caccttccgc ggcctctata tacactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accacggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagcggacta 300 cgttggggga tctggggctg gttcgacccc tggggccagg gcaccctggt caccgtctcc 360 tca 363 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-31 <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Val Ser Cys Lys Val Phe Gly Asp Thr Phe Arg Gly Leu             20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ser Gly Leu Arg Trp Gly Ile Trp Gly Trp Phe Asp Pro Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 19 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-31 <400> 19 gaaattgtgt tgacgcagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtattggc aacagcttag cctggtacca gcagaaacct 120 ggccaggctc ccaggctcct catgtatggt gcatccagca gggccactgg catcccagac 180 aggttcagtg gcagtggggc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg caacgtatta ctgtcagcag catactatcc caacattctc tttcggccct 300 gggaccaaag tggaagtcaa a 321 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-31 <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asn Ser             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Met         35 40 45 Tyr Gly Ala Ser Ser Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly     50 55 60 Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Thr Ile Pro Thr Phe                 85 90 95 Ser Phe Gly Pro Gly Thr Lys Val Glu Val Lys             100 105 <210> 21 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-32 <400> 21 gaggtgcagc tggtgcagtc tggggctgag ctgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttttggagg caccttcagt gacaatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatggggggc atcattccta tctttggaaa accaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cactgcctac 240 atggtcctga gcagcctgag atctgaggac acggccgtat attactgtgc gagaactatg 300 gttcggggct ttcttggggt tatggacgtc tggggccaag ggaccacggt caccgtctcc 360 tca 363 <210> 22 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-32 <400> 22 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe Ser Asp Asn             20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met         35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Val Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Thr Met Val Arg Gly Phe Leu Gly Val Met Asp Val Trp Gly             100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 23 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-32 <400> 23 gatattgtga tgacccagac tccatccttc ctgtccgcat ccataggaga cagagtcacc 60 atcacttgcc gggccagtca gggcattggc agttatttag cctggtatca gcaaagacca 120 ggggaagccc ctaagctcct gatctatgct gcatcgactt tgcaaagtgg agtcccatca 180 aggttcagcg gcagtggatc tgggacggac ttcactctca caatcagcaa cctgcagcct 240 gaagattttg caacttatta ctgtcaacag cttaataatt acccgatcac cttcggccaa 300 gggacacgac tggagattaa a 321 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-32 <400> 24 Asp Ile Val Met Thr Gln Thr Pro Ser Phe Leu Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Asn Tyr Pro Ile                 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 25 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-38 <400> 25 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcag 300 ttcgttacga tttttggagt gccaagatac ggtatggacg tctggggcca agggaccacg 360 gtcaccgtct cctca 375 <210> 26 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-38 <400> 26 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Asp Gln Phe Val Thr Ile Phe Gly Val Pro Arg Tyr Gly Met             100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 125 <210> 27 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-38 <400> 27 cagtctgccc tgactcagcc accctcagtg tccgtgtccc caggacagac agccaacatc 60 ccctgctctg gagataaatt ggggaataaa tatgcttact ggtatcagca gaagccaggc 120 cagtcccctg tactgctcat ctatcaagat atcaagcggc cctcaaggat ccctgagcga 180 ttctctggct ccaactctgc ggacacagcc actctgacca tcagcgggac ccaggctatg 240 gatgaggctg actattactg tcagacgtgg gacaacagcg tggtcttcgg cggcgggacc 300 aagctgaccg tcctc 315 <210> 28 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-38 <400> 28 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Asn Ile Pro Cys Ser Gly Asp Lys Leu Gly Asn Lys Tyr Ala             20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Leu Ile Tyr         35 40 45 Gln Asp Ile Lys Arg Pro Ser Arg Ile Pro Glu Arg Phe Ser Gly Ser     50 55 60 Asn Ser Ala Asp Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Asn Ser Val Val Phe                 85 90 95 Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 <210> 29 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-42 <400> 29 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtct attactgtgc gagagggcgt 300 caaatgttcg gtgcgggaat tgatttctgg ggcccgggca ccctggtcac cgtctcctca 360 <210> 30 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-42 <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr             20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe Trp Gly Pro             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 31 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-42 <400> 31 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgac agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagcg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcaa caatcgtcat 300 gtgatattcg gcggagggac caagctgacc gtccta 336 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-42 <400> 32 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn             20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val         35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser     50 55 60 Gly Ser Ile Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser                 85 90 95 Asn Asn Arg His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 33 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-46 <400> 33 gggtgcagc tggtggagtc tggggctgaa gtaaagaagc ctgggtcctc ggtgaaagtc 60 tcctgcaagg tttcaggagg cacattcggc acctatgctc tcaactgggt gcgccaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcgtccctc tcattggtct agtaaactac 180 gcacataact ttgagggcag aatctcgatt accgcggaca agtccacggg cacagcctac 240 atggaactga gcaacctgag atctgacgac acggccgtgt attactgtgc gagagaggtc 300 tacggtggta actccgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 34 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-46 <400> 34 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Gly Thr Phe Gly Thr Tyr             20 25 30 Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Val Pro Leu Ile Gly Leu Val Asn Tyr Ala His Asn Phe     50 55 60 Glu Gly Arg Ile Ser Ile Thr Ala Asp Lys Ser Thr Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Val Tyr Gly Gly Asn Ser Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 35 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-46 <400> 35 aattttatgc tgactcagcc ccactcagtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcactc gcagtagtgg caacattggc accaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccgtcgc tttgatctac gaggattatc gaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcat catctctgga 240 ctgaagcctg aggacgaggc tgactactac tgtcagtctt atcatagcag cggttgggaa 300 ttcggcggag ggaccaagct gaccgtcctc 330 <210> 36 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-46 <400> 36 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Asn Ile Gly Thr Asn             20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Val Ala Leu         35 40 45 Ile Tyr Glu Asp Tyr Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser     50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ser Ser I Le Ser Ser Ser 65 70 75 80 Leu Lys Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr His Ser                 85 90 95 Ser Gly Trp Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 37 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-50 <400> 37 caggtgcagc tggtgcagtc tggaggtgag gtgaagaagc cgggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttgagc agtcatggta taacctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctc acaatggtca cgctagcaat 180 gcacagaagg tggaggacag agtcactatg actactgaca catccacgaa cacagcctac 240 atggaactga ggagcctgac agctgacgac acggccgtgt attactgtgc gagagtacat 300 gctgccctct actatggtat ggacgtctgg ggccaaggaa ccctggtcac cgtctcctca 360 <210> 38 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-50 <400> 38 Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Ser His             20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Ser Ala His Asn Gly His Ala Ser Asn Ala Gln Lys Val     50 55 60 Glu Asp Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ala Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Val Ala Ala Leu Tyr Tyr Gly Met Asp Val Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 39 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-50 <400> 39 cagtctgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa ggtgtgcact ggtatcagca gaagccaggc 120 caggcccctg tactggtcgt ctatgatgat agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcattg ggtgttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 40 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-50 <400> 40 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Gly Val             20 25 30 His Trp Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr         35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser     50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His                 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 <210> 41 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-52 <400> 41 caggtgcagc tgcaggagtc ggggggaggc gtggtgcagc ctgggaggtc cctgagactc 60 tcctgttcag cctctggatt caccttcagc agacatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtg atatcacatg atggaagtgt aaaatactat 180 gcagactcca tgaagggccg attcagcatc tccagagaca attccaacaa cacactgtat 240 ctccaaatgg acagcctgag agctgacgac acggccgttt attactgtgc gagaggactg 300 tcgtaccagg tgtcggggtg gttcgacccc tggggccagg gcaccctggt caccgtctcc 360 tca 363 <210> 42 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-52 <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Arg His             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Ser His Asp Gly Ser Val Lys Tyr Tyr Ala Asp Ser Met     50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Asn Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Leu Ser Tyr Gln Val Ser Gly Trp Phe Asp Pro Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 43 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-52 <400> 43 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcac caccccttcg 300 gtgttcggcg gcgggaccaa gctgaccgtc cta 333 <210> 44 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-52 <400> 44 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn             20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val         35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser     50 55 60 Gly Ser Ile Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser                 85 90 95 Thr Thr Pro Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 45 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-55 <400> 45 caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg accagccctc ataatggtct cacagcattt 180 gcacagatcc tagagggccg agtcaccatg accacagaca catccacgaa cacagcctac 240 atggaattga ggaacctgac atttgatgac acggccgttt atttctgtgc gaaagtacat 300 cctgtcttct cttatgcgtt ggacgtctgg ggccaaggca ccctggtcac cgtctcctca 360 <210> 46 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-55 <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Thr Ser Pro His Asn Gly Leu Thr Ala Phe Ala Gln Ile Leu     50 55 60 Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Asn Leu Thr Phe Asp Asp Thr Ala Val Tyr Phe Cys                 85 90 95 Ala Lys Val His Pro Val Phe Ser Tyr Ala Leu Asp Val Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 47 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-55 <400> 47 aattttatgc tgactcagcc ccactctgtg tcggagtccc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaactatg tacagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtgatctat gaagataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacacctcc tccaactctg cctccctcac catctctgga 240 ctgaagacta aggacgaggc ggactactac tgtcagtctt atgatggcat cactgtgatt 300 ttcggcggag ggaccaagtt gaccgtccta 330 <210> 48 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-55 <400> 48 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn             20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val         35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser     50 55 60 Gly Ser Ile Asp Thr Ser Ser Asn Ser Ser Ser Ser Ser Ser Ser Ser Ser 65 70 75 80 Leu Lys Thr Lys Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly                 85 90 95 Ile Thr Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 49 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-56 <400> 49 gggtgcagc tggtggagtc tggagctgag gtgatgaacc ctgggtcctc ggtgagggtc 60 tcctgcaggg gttctggagg cgacttcagt acctatgctt tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat agcaaactac 180 gcacagaagt tccagggcag ggtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgacgat acggccgtgt attactgtgc gagagatggc 300 tatggttcgg acccggtgct atggggccag ggcaccctgg tcaccgtctc ctca 354 <210> 50 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-56 <400> 50 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Met Asn Pro Gly Ser 1 5 10 15 Ser Val Arg Ser Val Ser Cys Arg Gly Ser Gly Gly Asp Phe Ser Thr Tyr             20 25 30 Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Tyr Gly Ser Asp Pro Val Leu Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 51 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-56 <400> 51 aattttatgc tgactcagcc ccactctgtg tcggggtctc cggggaagac ggtaaccctc 60 ccctgcaccc gcagcagtgg cagcattgcc agccactatg tccagtggta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataaca agagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcag catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcag caatcgttgg 300 gtgttcggcg gagggaccaa gctgaccgtc cta 333 <210> 52 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-56 <400> 52 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Leu Pro Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser His             20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val         35 40 45 Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser     50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ser Ser Ser Ser Ser Ser Ser 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser                 85 90 95 Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 53 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-65 <400> 53 gaggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc aactatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagg tccagggcag agtcaccatg accacagaca catccacgag cacaggctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagaggagat 300 tttcggaaac cctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 54 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Variable heavy chain of Ab-65 <400> 54 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr             20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val     50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Gly Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Asp Phe Arg Lys Pro Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 55 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> Variable light chain of Ab-65 <400> 55 ctgcctgtgc tgactcagcc ggcttccctc tctgcatccc ccggagcatc agccagtctc 60 acctgcacct tacgcagtgg cctcaatgtt ggttcctaca ggatatactg gtaccagcag 120 aagccaggga gtcgtcccca gtatctcctg aactacaaat cagactcaaa taaacagcag 180 gcctctggag tccccagccg cttctctgga tccaaggatg cttcggccaa tgcagggatt 240 ttactcatct ccgggctcca gtctgaggat gaggctgact attactgtat gatttggtac 300 agcagcgctg tggtattcgg cggagggacc aagctgaccg tccta 345 <210> 56 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Variable light chain of Ab-65 <400> 56 Leu Pro Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Leu Asn Val Gly Ser             20 25 30 Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Arg Pro Gln Tyr         35 40 45 Leu Leu Asn Tyr Lys Ser Asp Ser Asn Lys Gln Gln Ala Ser Gly Val     50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile 65 70 75 80 Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys                 85 90 95 Met Ile Trp Tyr Ser Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu             100 105 110 Thr Val Leu         115 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 consensus, Ab-42 <400> 57 Ser Tyr Ala Ile Ser 1 5 <210> 58 <211> 5 <212> PRT <213> Artificial Sequence <220> Heavy chain CDR1 of Ab-14, Ab-55 <400> 58 Ser Tyr Gly Ile Ser 1 5 <210> 59 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-16 <400> 59 Ser Tyr Ala Leu Ser 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-22 <400> 60 Asp Tyr Ala Met His 1 5 <210> 61 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-30 <400> 61 Asn Tyr Asp Met Ser 1 5 <210> 62 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-31 <400> 62 Gly Leu Tyr Ile His 1 5 <210> 63 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-32 <400> 63 Asp Asn Ala Ile Ser 1 5 <210> 64 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-38 <400> 64 Ser Tyr Ala Met Ser 1 5 <210> 65 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-46 <400> 65 Thr Tyr Ala Leu Asn 1 5 <210> 66 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-50 <400> 66 Ser His Gly Ile Thr 1 5 <210> 67 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 Ab-52 <400> 67 Arg His Gly Met His 1 5 <210> 68 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-56 <400> 68 Thr Tyr Ala Phe Ser 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 of Ab-65 <400> 69 Asn Tyr Gly Ile Ser 1 5 <210> 70 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 consensus <400> 70 Trp Ile Ser Pro Ile Gly Gly Ser Thr Asn Tyr Ala Gln Lys Val Gln 1 5 10 15 Gly      <210> 71 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-14 <400> 71 Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Glu 1 5 10 15 Asp      <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-16 <400> 72 Ala Ile Ser Gly Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp      <210> 73 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-22 <400> 73 Leu Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp      <210> 74 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-30 <400> 74 Arg Val Asn Trp Asn Gly Gly Ser Thr Thr Tyr Ala Asp Ala Val Lys 1 5 10 15 Asp      <210> 75 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-31 <400> 75 Trp Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp      <210> 76 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-32 <400> 76 Trp Ile Ile Pro Ile Phe Gly Lys Pro Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp      <210> 77 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-38 <400> 77 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp      <210> 78 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-42 <400> 78 Trp Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp      <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-46 <400> 79 Arg Ile Val Pro Leu Ile Gly Leu Val Asn Tyr Ala His Asn Phe Glu 1 5 10 15 Asp      <210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-50 <400> 80 Trp Ile Ser Ala His Asn Gly His Ala Ser Asn Ala Gln Lys Val Glu 1 5 10 15 Asp      <210> 81 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-52 <400> 81 Val Ile Ser His Asp Gly Ser Val Lys Tyr Tyr Ala Asp Ser Met Lys 1 5 10 15 Asp      <210> 82 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-55 <400> 82 Trp Thr Ser Pro His Asn Gly Leu Thr Ala Phe Ala Gln Ile Leu Glu 1 5 10 15 Asp      <210> 83 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-56 <400> 83 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Glu 1 5 10 15 Asp      <210> 84 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 of Ab-65 <400> 84 Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val Glu 1 5 10 15 Asp      <210> 85 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 consensus <220> <221> misc_feature <222> (3) (17) <223> Xaa can be any naturally occurring amino acid <400> 85 Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Asp Val              <210> 86 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-14 <400> 86 Ala Leu Pro Ser Gly Thr Ile Leu Val Gly Gly Trp Phe Asp Pro 1 5 10 15 <210> 87 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-16 <400> 87 Asp Val Phe Pro Glu Thr Phe Ser Met Asn Tyr Gly Met Asp Val 1 5 10 15 <210> 88 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-22 <400> 88 Val Leu Leu Pro Cys Ser Ser Thr Ser Cys Tyr Gly Ser Val Gly Ala 1 5 10 15 Phe Asp Ile              <210> 89 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-30 <400> 89 Glu Phe Val Gly Ala Tyr Asp Leu 1 5 <210> 90 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-31 <400> 90 Gly Leu Arg Trp Gly Ile Trp Gly Trp Phe Asp Pro 1 5 10 <210> 91 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-32 <400> 91 Thr Met Val Arg Gly Phe Leu Gly Val Met Asp Val 1 5 10 <210> 92 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-38 <400> 92 Asp Gln Phe Val Thr Ile Phe Gly Val Pro Arg Tyr Gly Met Asp Val 1 5 10 15 <210> 93 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-42 <400> 93 Gly Arg Gln Met Phe Gly Ala Gly Ile Asp Phe 1 5 10 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-46 <400> 94 Glu Val Tyr Gly Gly Asn Ser Asp Tyr 1 5 <210> 95 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-50 <400> 95 Val His Ala Ala Leu Tyr Tyr Gly Met Asp Val 1 5 10 <210> 96 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-52 <400> 96 Gly Leu Ser Tyr Gln Val Ser Gly Trp Phe Asp Pro 1 5 10 <210> 97 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-55 <400> 97 Val His Pro Val Phe Ser Tyr Ala Leu Asp Val 1 5 10 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-56 <400> 98 Asp Gly Tyr Gly Ser Asp Pro Val Leu 1 5 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 of Ab-65 <400> 99 Gly Asp Phe Arg Lys Pro Phe Asp Tyr 1 5 <210> 100 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 consensus <400> 100 Thr Arg Ser Ser Gly Ser Ile Gly Ser Asn Tyr Val Gln 1 5 10 <210> 101 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-14 <400> 101 Thr Arg Ser Ser Gly Asn Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 102 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-16 <400> 102 Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 1 5 10 <210> 103 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-22 <400> 103 Gly Gly Ser Asp Ile Gly Arg Lys Ser Val His 1 5 10 <210> 104 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-30 <400> 104 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 105 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-31 <400> 105 Arg Ala Ser Gln Ser Ile Gly Asn Ser Leu Ala 1 5 10 <210> 106 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-32 <400> 106 Arg Ala Ser Gln Gly Ile Gly Ser Tyr Leu Ala 1 5 10 <210> 107 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-38 <400> 107 Ser Gly Asp Lys Leu Gly Asn Lys Tyr Ala Tyr 1 5 10 <210> 108 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-42 <400> 108 Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn Tyr Val Gln 1 5 10 <210> 109 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-46 <400> 109 Thr Arg Ser Ser Gly Asn Ile Gly Thr Asn Tyr Val Gln 1 5 10 <210> 110 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-50 <400> 110 Gly Gly Asn Asn Ile Gly Ser Lys Gly Val His 1 5 10 <210> 111 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-52 <400> 111 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 112 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-55 <400> 112 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 113 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-56 <400> 113 Thr Arg Ser Ser Gly Ser Ile Ala Ser His Tyr Val Gln 1 5 10 <210> 114 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 of Ab-65 <400> 114 Thr Leu Arg Ser Gly Leu Asn Val Gly Ser Tyr Arg Ile Tyr 1 5 10 <210> 115 <211> 7 <212> PRT <213> Artificial Sequence <220> Light chain CDR2 of consensus, Ab-14, Ab-42, Ab-52, Ab-55 <400> 115 Glu Asp Asn Gln Arg Pro Ser 1 5 <210> 116 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-16 <400> 116 Gly Lys Asn Asn Arg Pro Ser 1 5 <210> 117 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-22 <400> 117 Ser Asp Arg Asp Arg Pro Ser 1 5 <210> 118 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-30 <400> 118 Asp Val Ser Asn Arg Pro Ser 1 5 <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-31 <400> 119 Gly Ala Ser Ser Ala Thr 1 5 <210> 120 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-32 <400> 120 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 121 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-38 <400> 121 Gln Asp Ile Lys Arg Pro Ser 1 5 <210> 122 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-46 <400> 122 Glu Asp Tyr Arg Arg Pro Ser 1 5 <210> 123 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-50 <400> 123 Asp Asp Ser Asp Arg Pro Ser 1 5 <210> 124 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-56 <400> 124 Glu Asp Asn Lys Arg Pro Ser 1 5 <210> 125 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 of Ab-65 <400> 125 Tyr Lys Ser Asp Ser Asn Lys Gln Gln Ala Ser 1 5 10 <210> 126 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 consensus <400> 126 Gln Ser Tyr Asp Ser Ser Thr Trp Val 1 5 <210> 127 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-14 <400> 127 Gln Ser Tyr Asp Ser Ser Asn Leu Trp Val 1 5 10 <210> 128 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-16 <400> 128 Asn Ser Arg Asp Ser Ser Gly Asn His Tyr Val 1 5 10 <210> 129 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-22 <400> 129 Gln Val Trp Asp Asn Asn Ser Asp His Tyr Val 1 5 10 <210> 130 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-30 <400> 130 Ser Ser Tyr Thr Ser Ser Thr Leu Pro 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-31 <400> 131 Gln Gln His Thr Ile Pro Thr Phe Ser 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-32 <400> 132 Gln Gln Leu Asn Asn Tyr Pro Ile Thr 1 5 <210> 133 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-38 <400> 133 Gln Thr Trp Asp Asn Ser Val Val 1 5 <210> 134 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-42 <400> 134 Gln Ser Tyr Asp Ser Asn Asn Arg His Val Ile 1 5 10 <210> 135 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-46 <400> 135 Gln Ser Tyr His Ser Ser Gly Trp Glu 1 5 <210> 136 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-50 <400> 136 Gln Val Trp Asp Ser Ser Ser Asp His Trp Val 1 5 10 <210> 137 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-52 <400> 137 Gln Ser Tyr Asp Ser Thr Thr Pro Ser Val 1 5 10 <210> 138 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-55 <400> 138 Gln Ser Tyr Asp Gly Ile Thr Val Ile 1 5 <210> 139 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-56 <400> 139 Gln Ser Tyr Asp Ser Ser Asn Arg Trp Val 1 5 10 <210> 140 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 of Ab-65 <400> 140 Met Ile Trp Tyr Ser Ser Ala Val Val 1 5

Claims (36)

As isolated humanized monoclonal antibodies,
The heavy chain having three CDRs each comprising the amino acid sequences SYAIS (SEQ ID NO: 57), GIIPIFGTANYAQKFQG (SEQ ID NO: 78) and GRQMFGAGIDF (SEQ ID NO: 93), and the amino acid sequences TRSSGSIDSNYVQ (SEQ ID NO: 108), EDNQRPS Lt; RTI ID = 0.0 &gt; CDRs &lt; / RTI &gt; comprising QSYDSINNRHVI (SEQ ID NO: 134)
Wherein said antibody binds to a human prospective ligand-1. The antibody of claim 1, wherein the antibody is monovalent or divalent. The antibody of claim 1, wherein said antibody is a single chain antibody. The antibody of claim 1, wherein said antibody is a bispecific antibody that also binds to a tumor-associated antigen, cytokine, or cell surface receptor. 5. The antibody of claim 4, wherein the tumor-associated antigen is CAIX. 5. The antibody of claim 4, wherein said cytokine is IL-10. 5. The antibody of claim 4, wherein said cell surface receptor is CCR4, IL21R, BTLA, HVEM or TIM3. A single-chain antibody encoded by a nucleotide sequence comprising a V _H nucleotide sequence comprising SEQ ID NO: 29 and a V _L nucleotide sequence comprising SEQ ID NO: 31, wherein the antibody binds to a human prospective ligand-1. 34. A single chain antibody comprising a V _H amino acid sequence comprising SEQ ID NO: 30 and a V _L amino acid sequence comprising SEQ ID NO: 32, wherein the antibody binds to a human prospective ligand-1. 10. The antibody of any one of claims 1 to 9, wherein said antibody binds to a therapeutic agent. 11. The antibody of claim 10, wherein said therapeutic agent comprises a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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